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Session Schedule & Abstracts




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Saturday 2nd July, 2016

DCT2
Symposium: Diffusible iodine-based contrast-enhanced computed tomography (diceCT) and related imaging techniques for evolutionary morphology 2

Room: Salon A   11:30 am–1:00 pm

Moderator(s): P. M. Gignac, A. N. Herdina, N. J. Kley, A. Morhardt, J. A. Clarke, & M. Colbert
DCT2-1  11:30 am  Musculoskeletal modelling and simulations of the mouse hindlimb during locomotion: the role of high-resolution scanning and contrast imaging. Charles JP*, Royal Veterinary College; Cappellari O, Royal Veterinary College; Spence AJ, Temple University ; Wells DJ, Royal Veterinary College; Hutchinson JR, Royal Veterinary College   jcharles@rvc.ac.uk
Abstract: Terrestrial animals are able to move over various terrains in a stable and controlled way. This occurs through interactions between nerves, muscles and the environment, however their inherent complexity means they are not yet understood. Here we describe the creation of a biomechanical model of the hindlimb of the mouse, an animal commonly used in studies related to treatments for neuromuscular disorders and movement control. With this model, it will be possible to explore the intricacies of vertebrate locomotion in new detail, and as mice are thought to be close to the morphology of early mammals, it may give insights into the evolution of this lineage. To develop the model, 39 muscles of the hindlimb were identified through I2KI enhanced microCT scanning, which allowed muscle attachment points to be determined. These were then dissected to determine their architecture, which was used to estimate their force-generating and length-change potential. A sensitivity analysis supported its validity. To simulate mouse locomotion, the hindlimb kinematics and ground reaction forces throughout a single representative stride were measured experimentally and added to the model. It was then possible to calculate individual muscle moments around each joint as well as predict patterns of muscle activation during running. Using a forward dynamics approach, we can predict the responses within the hindlimb to sensory or motor perturbations. These responses will be compared to experimental data, where optogenetics will be used to apply perturbations during movement. This systems approach may give valuable and novel insights into neuromuscular movement control, both within small rodents and potentially more generally within terrestrial vertebrates. Importantly, all of this potential for biomechanical simulation and insight into neuromotor control within animals such as the mouse depends on the high-fidelity imaging of musculoskeletal anatomy enabled by I2KI enhanced microCT scanning.

DCT2-2  11:45 am  The evolution of the mammalian jaw adductor musculature—inferences from soft-tissue imaging of extant taxa. Lautenschlager S*, University of Bristol, School of Earth Sciences; Gill P, University of Bristol, School of Earth Sciences; Fagan M, University of Hull, School of Engineering; Rayfield E J, University of Bristol, School of Earth Sciences   glzsl@bristol.ac.uk
Abstract: The evolution of the mammalian jaw is characterised by the gradual reduction of its individual bones into a single element and the concomitant transformation of the jaw joint and incorporation of the post-dentary bones into the middle ear complex. This osteological transformation is accompanied by a rearrangement and modification of the jaw adductor musculature, which is thought to have allowed the evolution of a more efficient masticatory system in comparison to the plesiomorphic reptilian condition. While osteological characters relating to this transition are well documented in the fossil record, little is known about the exact arrangement of the individual adductor muscles and reconstructions have often been unclear or conjectural. Here, we use comparative data derived from contrast-enhanced CT scanning of an extant taxon (e.g. Monodelphis) and digital restoration techniques to reconstruct the jaw adductor musculature of a number of representative non-mammalian cynodonts and mammaliaforms (including Thrinaxodon, Diademodon, Probainognathus, Morganucodon). Three-dimensional digital models of the adductor muscles were created on the basis of osteological correlates, homological criteria and spatial constraints. Different hypothesized arrangements were tested taking into account maximum muscle stretch factors and differences in muscle architecture. Results of this study show that the mammalian muscle division, including a loss of the pseudotemporalis musculature and the separation of the masseter, was already present in Eucynodontia. Furthermore, the mammalian muscle arrangement, with a shift of the pterygoideus musculature to the dentary, was completed in early mammaliaforms. Consequently, both events appear to have preceded the formation of the temporomandibular jaw joint and the appearance of true mammals.

DCT2-3  12:00 pm  Masticatory muscle anatomy of African mole-rats revealed by diceCT. Cox PG*, University of York   philip.cox@hyms.ac.uk
Abstract: African mole-rats (Bathyergidae) are a family of rodents united by a subterranean lifestyle. Of the six extant genera of mole-rats, five are known to construct their burrows using chisel-tooth digging i.e. they dig with their incisors. The remaining genus, Bathyergus, is a scratch digger, tunnelling using its forelimbs only. Previous research has shown that the chisel-tooth digging bathyergids all show cranial and mandibular morphologies that facilitate high bite force and wide gape. However, much less is known about the morphology of the masticatory musculature in mole-rats, and whether differences exist between the scratch and chisel-tooth diggers in the size or configuration of the muscles. Diffusible iodine-based contrast enhanced CT (diceCT) provides a method to visualise soft tissues, such as muscles, with CT scanning. Six specimens of mole-rat heads, one representing each extant genus, were fixed in formaldehyde and stained with a 3.75% solution of iodine-potassium iodide for several months, before being scanned with microCT. Voxel dimensions of the resulting image stacks ranged from 0.04-0.08 mm. The scans revealed a highly complex set of jaw-closing muscles in the Bathyergidae. The masseter complex is the largest component of the adductor musculature, forming 50-60% of the total muscle mass. Unlike other families in the Ctenohystrica, mole-rats also have a large temporalis muscle, comprising 25-30% of muscle mass. Differences between the chisel-tooth and scratch diggers are largely seen in the size of the pterygoid muscles, which are highly reduced in Bathyergus compared to the other mole-rats. However, despite minor variations, the relative sizes of the jaw-closing muscles in the Bathyergidae are remarkably consistent across species. Overall, it appears that differences in bite-force abilities between mole-rat genera are attributable to variations in the orientation of muscle pull rather than muscle mass.

DCT2-4  12:15 pm  Integration of diceCT with XROMM and fluoromicrometry enhances functional morphology and biomechanics research: a case study of the macaque (Mammalia: Primates) feeding apparatus. Orsbon CP*, University of Chicago; Gidmark NJ, University of Chicago; Ross CF, University of Chicago   orsbon@uchicago.edu
Abstract: X-ray Reconstruction of Moving Morphology (XROMM) and fluoromicrometry permit unprecedented opportunities to study functional morphology and biomechanics with high precision and in three-dimensions. Adaptation of these techniques to study muscle function often requires time-intensive dissection and measurement that destroy the specimen in order to confirm marker location and muscular attachment sites on bone. This research bypasses such destruction and presents the integration of diffusible iodine-based contrast-enhanced computed tomography (DiceCT) into our model system for the neuromechanics of primate feeding, which uses the rhesus macaque (Macaca mulatta) and employs XROMM, fluoromicrometry, electromyography, and nerve stimulation experiments. Specifically, we visualize soft tissue structures using DiceCT to reconstruct in vivo muscle length changes, confirm marker and electrode placement, and plan surgical approaches. A known drawback to staining involves loss of contrast between bone and other structures; therefore, specimens used for XROMM should have a prestaining scan in order obtain bone models. Alternative stains, such as phosphomolybdic acid (PMA), produce greater contrast between muscle and bone; however, PMA's slow diffusion rate, high cost, and permanent discoloration may be undesirable for those studying larger organisms. We register post-staining scans with pre-staining scans to superimpose soft tissue structures of interest, such as muscle and tendon attachments, onto the bones for reconstruction of soft tissue dynamics. We also use DiceCT to visualize regions with significant morphological complexity, such as the infratemporal fossa, where surgical targets for nerve stimulation experiments are imbedded in vascular structures. DiceCT gives researchers unprecedented insight into morphology, and its integration with XROMM and fluoromicrometry will enhance the impact of these methods on the fields of functional morphology and biomechanics.

DCT2-5  12:30 pm  Contrast-enhanced CT provides insight into amphibian lingual morphology. Stanley EL*, Florida Museum of Natural History; Blackburn DC, Florida Museum of Natural History   elstanley@flmnh.ufl.edu
Abstract: The tongue plays an important role in amphibian prey capture, and several lingual projection modes are known. Some amphibians utilize hydrostatic (frogs: Hemisus, Microhylidae) or ballistic (salamanders: Bolitoglossa, Hydromantes) tongue projection, though the most common method involves contracting the protractor muscles to cause the sticky tongue pad to flip forward and secure the prey. Newly developed imaging methodologies allow the structures of the tongue to be visualized and analyzed at high resolutions in three dimensions, expanding on the data available from traditional methods (histology, dissection) and allowing for larger-scale comparative surveys of tongue anatomy across amphibian diversity. Here we present processed data from diffusible iodine-based contrast-enhanced (dice) CT scans of ranoid frogs, reconstructing serial 2D tomograms and 3D volumes to visualize and quantify anuran lingual anatomy. We compare our findings with those of previous studies for the same taxa using histology and scanning electron microscopy, and provide insights into the relationship between the morphology of the tongue and other anatomical systems.

DCT2-6  12:45 pm  Studying metamorphosis of the cranial musculoskeletal system in the axolotl using contrast-enhanced µCT. Pardo J.D.*, University of Calgary; Shipclark R., University of Calgary; Szostakiwskyj M., University of Calgary; Anderson J.S., University of Calgary   jdpardo@ucalgary.ca
Abstract: In amphibians, metamorphosis is understood as rapid attainment of somatic maturity associated with the transition from water to land, but how developmental processes reorganize musculoskeletal morphology is only beginning to be understood. We used contrast-enhanced µCT to study changes in the cranial musculoskeletal system of the axolotl (Ambystoma mexicanum) across experimentally-induced metamorphosis. We identify significant changes in cranial osteology by 28 days post-injection, including reorganization of the palate and lower jaw, and expansion of some ossification centers in the palate, braincase, and cranial vault. Changes in the cranial skeleton are matched by changes in cranial musculature, including an increase in the size of the optic retractor, jaw depressor, and opercularis musculature. We identify a number of morphological systems to target for future studies of metamorphosis, and note differences in the impact of metamorphosis on neurocranial and dermatocranial ossifications. We identify a number of osteological features that, although previously conceptualized as evidence of paedomorphosis or miniaturization in lissamphibians, are instead indicative of exaggeration of late-ontogeny remodeling processes, with implications for lissamphibian phylogeny.



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