Micro-CT imaging of live insects using carbon dioxide gas-induced hypoxia as anesthetic with minimal impact on certain subsequent life history traits

Abstract Background Live imaging of whole invertebrates can be accomplished with X-ray micro-computed tomography (micro-CT) at 10-100 μm spatial resolution. However, image quality could be compromised by the movement of live subjects, producing image artefacts. We tested the feasibility of using CO2 gas to induce temporary full-immobilization of sufficient duration to image live insects based on their ability to tolerate hypoxic conditions. Additionally, we investigated the effects of these prolonged hypoxic conditions on several life history traits of a lepidopteran species. Methods Live Colorado potato beetle (CPB) and true armyworm (TAW) adults were immobilized under a constant CO2 gas flow (0.5 L/min), and scanned using micro-CT (80 kVp; 450 μA). An L8 (24) orthogonal array (OA) was used to evaluate the effects of prolonged CO2-induced anesthesia on the recovery, longevity, and incidence of mating of TAW adults. The variable factors were age (immature and mature), sex (female and male), exposure time (3 and 7 h), and exposure regime (single and repeated). Results With this method, successful 3D reconstruction and visualizations of CPB and TAW adults were produced at 20 micron voxel spacing at an acceptable radiation dose and image noise level. From the inverse-square relationship found between the radiation doses and image noise levels, the optimal scanning protocol produced an entrance dose of 6.2 ± 0.04 Gy with images of 129.6 ± 5.1 HU noise level during a 2.7 h scan. Independent OA experiments indicated that CO2 gas did not result in death of exposed TAW adults, except when older males were exposed for longer durations. Exposure time and sex were more influential factors affecting recovery, longevity, and mating success than age and exposure regime following CO2 exposure. Conclusion We have demonstrated that using CO2 gas during micro-CT imaging effectively induces safe, repeatable, whole-body, and temporary immobilization of live insects for 3D visualizations without motion artefacts. Moreover, we have shown that exposed TAW individuals made a full recovery with very little impact on subsequent longevity, and mating success post hypoxia. This method is applicable to other imaging modalities and could be used for routine exploratory and time-course studies, for repeated scanning of live and intact individuals

BRM Immunotherapy of Orthotopically Implanted Murine Bladder Tumours: Treatment Response by Monitoring MRI

The authors evaluated magnetic resonance imaging (MRI) for monitoring orthotopic bladder tumour growth and treatment response to intravesical immunotherapy with the biological response modifiers (BRMs): recombinant tumour necrosis factor alpha (TNF-α), combination of TNF-α plus interferon gamma (IFN-γ) and interleukin-2 (IL-2). MRI demonstrated detection of early superficial murine bladder tumour (MBT-2) and accurate sequential assessment of the topography and depth of intravesical tumour involvement. Response to intravesical instillations with multiple doses ofBRMs was assessed against early stage MBT-2 bladder tumours (confirmed by MRI) 14 days after transurethral tumour implantation. Serial MRI scans of TNF-α treated mice revealed significant retardation of tumour growth which correlated well with corresponding histological examination of the whole mount bladder sections illustrating areas and depth of tumour regression. Intravesical instillation of combination TNF-α plus IFN-γ into tumour-bearing mice caused tumour growth inhibition up to 21 days following treatment; the results, however, were not superior to those noted with TNF-α alone. Sequential MR images of tumour-bearing bladders following intravesical treatment with IL-2 revealed tumour regression with no visible tumour from day 21 to 33 post tumour implant. Histological examination revealed foci of carcinoma in situ only. Control untreated bladders revealed deeply invasive transitional cell carcinoma. These results show that MRI offers a dependable tool for noninvasive monitoring of tumour growth and of the course of experimental bladder tumour during therapy

Computed Blood Flow Dynamics In An Anatomically Realistic Cerebral Aneurysm

this paper we present a computational fluid dynamic (CFD) simulation of physiologically pulsatile flow in an anatomically realistic aneurysm reconstructed from in vivo 3-D imaging of a human subjec

Rapid in Vivo Whole Body Composition of Rats Using Cone Beam µCT

Precise, noninvasive analysis and quantification of in vivo body composition is essential for research involving longitudinal, small-animal disease models. We investigated the feasibility and precision of a rapid, flat-panel μCT scanner to report whole body adipose tissue volume (ATV), lean tissue volume (LTV), skeletal tissue volume (STV), and bone mineral content (BMC) in 25 postmortem female and 52 live male Sprague-Dawley rats. μCT images, acquired in three 90-mm segments and reconstructed with 308 μm of isotropic voxel spacing, formed contiguous image volumes of each entire rat specimen. Three signal-intensity thresholds (determined to be -186, 5, and 155 HU) were used to classify each voxel as adipose, lean, or skeletal tissue, respectively. Tissue masses from the volume fractions of ATV, LTV, and STV were calculated from assumed tissue densities of 0.95, 1.05, and 1.92 g/cm(-3), respectively. A CT-derived total mass was calculated for each rat and compared with the gravimetrically measured mass, which differed on average for the postmortem female and the live male group by 2.5 and 1.1%, respectively. To evaluate the accuracy of the CT-derived body composition technique, following the live male study excised muscle tissue in the lower right leg of all rats in group B were compared with the image-derived LT measurement of the same regional compartment and found to differ on average by 2.2%. Through repeated CT measurements of postmortem specimens, the whole body ATV, LTV, STV, and BMC measurement analysis gave a precision value of ±0.6, 1.9, 1.7, and 0.5% of the average value, respectively

Loss of the mammalian DREAM complex deregulates chondrocyte proliferation

Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes. © 2014, American Society for Microbiology