Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using <em>In Vivo</em> Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

Brock University

Adipose triglyceride lipase (ATGL) catalyzes the rate-limiting removal of the first fatty acid from a triglyceride. ATGL is activated by comparative gene identification-58 and inhibited by G(0)/G(1) switch gene-2 protein (G0S2). Research in other tissues and cell culture indicates that inhibition is dependent on relative G0S2-to-ATGL protein content. G0S2 may also have several roles within mitochondria; however, this has yet to be observed in skeletal muscle. The purpose of this study was to determine if muscle G0S2 relative to ATGL content would decrease to facilitate intramuscular lipolysis following endurance training. Male Sprague-Dawley rats (n = 10; age 51-53 days old) were progressively treadmill trained at a 10% incline for 8 wk ending with 25 m/min for 1 h compared with control. Sciatic nerve stimulation for hind-limb muscle contraction (and lipolysis) was administered for 30 min to one leg, leaving the opposing leg as a resting control. Soleus (SOL), red gastrocnemius (RG), and white gastrocnemius were excised from both legs following stimulation or control. ATGL protein increased in all trained muscles. Unexpectedly, G0S2 protein was greater in the trained SOL and RG. In RG-isolated mitochondria, G0S2 also increased with training, yet mitochondrial G0S2 content was unaltered with acute contraction; therefore, any role of G0S2 in the mitochondria does not appear to be acutely mediated by content alone. In summary, G0S2 increased with training in oxidative muscles and mitochondria but not following acute contraction, suggesting that inhibition is not through relative G0S2-to-ATGL content but through more complicated intracellular mechanisms.

Increases in skeletal muscle ATGL and its inhibitor G0S2 following 8 weeks of endurance training in metabolically different rat skeletal muscles.

In vivo micro-computed tomography (μCT) provides the ability to measure longitudinal changes to tibia microarchitecture, but the effect of this radiation is not well understood. The right proximal tibia of Sprague-Dawley rats (n = 12/group) randomized to Sham-control (Sham) or ovariectomy (OVX) surgery at 12 weeks of age was scanned using μCT at 13, 17, 21, and 25 weeks of age, at a resolution of 18 μm and a radiation dose of 603 mGy. The left proximal tibia was scanned only at 25 weeks of age to serve as an internal non-irradiated control. Repeated irradiation did not affect tibia microarchitecture in Sham or OVX groups, although there was an increase in cortical eccentricity (P < 0.05). All trabecular outcomes and cortical BMD were different (P < 0.05) between groups after only 1 week post-surgery and differences persisted to study endpoint. Characteristic changes to trabecular bone were observed in OVX rats over time. Interactions of time and hormone status were found for cortical BMD (P < 0.001), Ps. Pm., and Ec. Pm. (P < 0.05). Repeated irradiation of the tibia at 13, 17, 21, and 25 weeks does not cause adverse effects to microarchitecture, regardless of hormone status. This radiation dose can be applied over a typical 3-month study period to comprehensively understand how an intervention alters tibia microarchitecture without confounding effects of radiation.

Longitudinal Use of Micro-computed Tomography Does Not Alter Microarchitecture of the Proximal Tibia in Sham or Ovariectomized Sprague-Dawley Rats.

Osteoporosis is a global health problem that leads to an increased incidence of fragility fracture. Recent dietary patterns of Western populations include higher than recommended intakes of n-6 (ω-6) polyunsaturated fatty acids (PUFAs) relative to n-3 (ω-3) PUFAs that may result in a chronic state of sterile whole body inflammation. Findings from human bone cell culture experiments have revealed both benefits and detriments to bone-related outcomes depending on the quantity and source of PUFAs. Findings from observational and randomized controlled trials suggest that higher fatty fish intake is strongly linked with reduced risk of fragility fracture. Moreover, human studies largely support a greater intake of total PUFAs, total n-6 (ω-6) fatty acid, and total n-3 (ω-3) fatty acid for higher bone mineral density and reduced risk of fragility fracture. Less consistent evidence has been observed when investigating the role of long chain n-3 (ω-3) PUFAs or the ratio of n-6 (ω-6) PUFAs to n-3 (ω-3) PUFAs. Aspects to consider when interpreting the current literature involve participant characteristics, study duration, diet assessment tools, and the primary outcome measure.

PUFAs, Bone Mineral Density, and Fragility Fracture: Findings from Human Studies.

Bone microarchitecture, bone mineral density (BMD), and bone strength are affected positively by impact activities such as running; however, there are discrepancies in the magnitude of these effects. These inconsistencies are mainly a result of varying training protocols, analysis techniques, and whether or not the skeletal sites measured are weight bearing. This study's purpose was to determine the effects of endurance running on sites that experience different weight bearing and load. Eight-week-old male Sprague-Dawley rats (n = 20) were randomly assigned to either a group with a progressive treadmill running protocol (25 m/min for 1 h, incline of 10%) or a nontrained control group for 8 weeks. The trabecular structure of the tibia, lumbar vertebra (L3), and mandible and the cortical structure at the tibia midpoint were measured using microcomputed tomography to quantify bone volume fraction (i.e., bone volume divided by total volume (BV/TV)), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and cortical thickness. BMD at the proximal tibia, lumbar vertebrae (L1-L3), and mandible was measured using dual energy X-ray absorptiometry. The tibia midpoint strength was measured by 3-point bending using a materials testing system. Endurance running resulted in superior bone structure at the proximal tibia (12% greater BV/TV (p = 0.03), 14% greater Tb.N (p = 0.01), and 19% lower Tb.Sp (p = 0.05)) but not at other sites. Contrary to our hypothesis, mandible bone structure was altered after endurance training (8% lower BV/TV (p < 0.01) and 15% lower Tb.Th (p < 0.01)), which may be explained by a lower food intake, resulting in less mechanical loading from chewing. These results highlight the site-specific effects of loading on the skeleton.

Skeletal site-specific effects of endurance running on structure and strength of tibia, lumbar vertebrae, and mandible in male Sprague-Dawley rats.

The parameters of a micro-computed tomography (μCT) scan, including whether a bone is imaged in vivo or ex vivo, determine the quality of the resulting image. In turn, this impacts the accuracy of the trabecular and cortical outcomes. The absolute impact of μCT scanning at different voxel sizes and whether the sample is imaged in vivo or ex vivo on the morphological outcomes of the proximal tibia in the rat is unknown. The right proximal tibia of 6-month-old Sham-control and ovariectomized (OVX) rats (n = 8/group) was scanned using μCT (SkyScan 1176, Bruker, Kontich, Belgium) using three sets of parameters (9 μm ex vivo, 18 μm ex vivo, 18 μm in vivo) to compare the trabecular and cortical outcomes. Regardless of scan protocols, differences between Sham and OVX groups were observed as expected. At a voxel size of 18 μm, scanning in vivo or ex vivo had no effect on any of the outcomes measured. However, compared to a 9 μm voxel size scan, imaging at 18 μm resulted in significant underestimation of the connectivity density (p < 0.05) of the trabecular bone and a significant overestimation (p < 0.05) of the trabecular indices (trabecular thickness, degree of anisotropy) and of the cortical indices (cortical bone area, cortical area fraction, cortical thickness) in both Sham and OVX rats. These results suggest the benefit to scanning the proximal tibia of rats at a voxel size as low as 9 μm, although considerations must be made for the increased acquisition time, anesthesia, animal welfare, and radiation exposure associated with lower voxel size in vivo scanning. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1690-1698, 2017.

Comparison of ex vivo and in vivo micro-computed tomography of rat tibia at different scanning settings.

Higher intakes of polyunsaturated fatty acids (PUFA) are associated with benefits at several skeletal sites in postmenopausal women and in rodent models, but the effect of PUFA-containing oils on tooth-supporting alveolar bone of the mandible has not been studied. Moreover, direct comparison of the effect of flaxseed oil (a source of alpha-linolenic acid (ALA)) and menhaden oil (a source of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) is unknown. One-month old female Sprague-Dawley rats (n = 48) were randomized to and fed a diet containing flaxseed oil or menhaden oil from one to six months of age. At three months of age, rats were randomized to receive SHAM or ovariectomy (OVX) surgery (n = 12/diet). The inter-radicular septum below the first molar of the mandible was imaged at 6 months of age (study endpoint) using micro-computed tomography (μCT) at a resolution of 9 μm. As expected, OVX significantly reduced percent bone volume (BV/TV), connectivity density (Conn. D.), trabecular number (Tb. N.), and increased trabecular separation (Tb. Sp.) compared to SHAM rats (p < 0.001). However, post hoc analysis revealed these differences were present in rats fed menhaden oil but not those fed flaxseed oil. These results suggest that providing flaxseed oil, possibly through its high ALA content, provides protection against the OVX-induced alveolar bone loss in rats.

Providing Flaxseed Oil but Not Menhaden Oil Protects against OVX Induced Bone Loss in the Mandible of Sprague-Dawley Rats.

Skeletal health is a lifelong process impacted by environmental factors, including nutrient intake. The n-3 source and PUFA ratio affect bone health in growing rats, or following ovariectomy (OVX), but no study has investigated the longitudinal effect of PUFA-supplementation throughout these periods of bone development.

Lifelong intake of flaxseed or menhaden oil to provide varying n-6 to n-3 PUFA ratios modulate bone microarchitecture during growth, but not after OVX in Sprague-Dawley rats.

Long-term effects of repeated in vivo micro-computed tomography (μCT) scanning at key stages of growth and bone development (ages 2, 4 and 6 months) on trabecular and cortical bone structure, as well as developmental patterns, have not been studied. We determined the effect of repetitive μCT scanning at age 2, 4 and 6 months on tibia bone structure of male and female CD-1 mice and characterized developmental changes. At 2, 4 and 6 months of age, right tibias were scanned using in vivo μCT (Skyscan 1176) at one of three doses of radiation per scan: 222, 261 or 460 mGy. Left tibias of the same mice were scanned only at 6 months to serve as non-irradiated controls to determine whether recurrent radiation exposure alters trabecular and cortical bone structure at the proximal tibia. In males, eccentricity was lower (P<0.05) in irradiated compared with non-irradiated tibias (222 mGy group). Within each sex, all other structural outcomes were similar between irradiated and non-irradiated tibias regardless of dose. Trabecular bone loss occurred in all mice due to age while cortical development continued to age 6 months. In conclusion, repetitive μCT scans at various radiation doses did not damage trabecular or cortical bone structure of proximal tibia in male and female CD-1 mice. Moreover, scanning at 2, 4 and 6 months of age highlight the different developmental time course between trabecular and cortical bone. These scanning protocols can be used to investigate longitudinal responses of bone structures to an intervention.

Repeated irradiation from micro-computed tomography scanning at 2, 4 and 6 months of age does not induce damage to tibial bone microstructure in male and female CD-1 mice.

Amanda B. Longo

Department of Kinesiology,
Faculty of Applied Health Sciences,
Department of Kinesiology, Faculty of Applied Health Sciences

Brock University

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

Amanda B. Longo

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Kinesiology,Faculty of Applied Health Sciences,Department of Kinesiology, Faculty of Applied Health Sciences

Brock University

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

Department of Kinesiology,
Faculty of Applied Health Sciences,
Department of Kinesiology, Faculty of Applied Health Sciences