Suzanne L. Duce

Multiwall carbon nanotubes as MRI contrast agents for tracking stem cells

In this study we investigate the potential of multiwall carbon nanotubes (MWCNTs) with low metal impurities (2.57% iron) as magnetic resonance imaging (MRI) contrast agents. Taking into account probable aggregation at high MWCNTs concentration analysis shows that the r(2) relaxivity of MWCNTs in 1% agarose gels at 19 °C is 564 ± 41 s(-1) mM(-1); this is attributed to both the presence of iron oxide impurities and also to the carbon MWCNT structure itself. Stem cells were labelled with MWCNTs to demonstrate the effectiveness of MWCNTs as MRI contrast agents for cellular MRI. The MWCNTs did not impair cell viability or proliferation. These results suggest that the MRI contrast agent properties of the MWCNTs could be used in vivo for stem cell tracking/imaging and during MWCNT-mediated targeted electro-chemotherapy of tumours.

Micro-magnetic resonance imaging of avian embryos

Chick embryos are useful models for probing developmental mechanisms including those involved in organogenesis. In addition to classic embryological manipulations, it is possible to test the function of molecules and genes while the embryo remains within the egg. Here we define conditions for imaging chick embryo anatomy and for visualising living quail embryos. We focus on the developing limb and describe how different tissues can be imaged using micro‐magnetic resonance imaging and this information then synthesised, using a three‐dimensional visualisation package, into detailed anatomy. We illustrate the potential for micro‐magnetic resonance imaging to analyse phenotypic changes following chick limb manipulation. The work with the living quail embryos lays the foundations for using micro‐magnetic resonance imaging as an experimental tool to follow the consequences of such manipulations over time.

3D MRI Analysis of the Lower Legs of Treated Idiopathic Congenital Talipes Equinovarus (Clubfoot)

Background Idiopathic congenital talipes equinovarus (CTEV) is the commonest form of clubfoot. Its exact cause is unknown, although it is related to limb development. The aim of this study was to quantify the anatomy of the muscle, subcutaneous fat, tibia, fibula and arteries in the lower legs of teenagers and young adults with CTEV using 3D magnetic resonance imaging (MRI), and thus to investigate the anatomical differences between CTEV participants and controls. Methodology/Principal Findings The lower legs of six CTEV (2 bilateral, 4 unilateral) and five control young adults (age 12–28) were imaged using a 3T MRI Philips scanner. 5 of the CTEV participants had undergone soft-tissue and capsular release surgery. 3D T1-weighted and 3D magnetic resonance angiography (MRA) images were acquired. Segmentation software was used for volumetric, anatomical and image analysis. Kolmogorov-Smirnov tests were performed. The volumes of the lower affected leg, muscle, tibia and fibula in unilateral CTEV participants were consistently smaller compared to their contralateral unaffected leg, this was most pronounced in muscle. The proportion of muscle in affected CTEV legs was significantly reduced compared with control and unaffected CTEV legs, whilst proportion of muscular fat increased. No spatial abnormalities in the location or branching of arteries were detected, but hypoplastic anomalies were observed. Conclusions/Significance Combining 3D MRI and MRA is effective for quantitatively characterizing CTEV anatomy. Reduction in leg muscle volume appears to be a sensitive marker. Since 5/6 CTEV cases had soft-tissue surgery, further work is required to confirm that the treatment did not affect the MRI features observed. We propose that the proportion of muscle and intra-muscular fat within the lower leg could provide a valuable addition to current clinical CTEV classification. These measures could be useful for clinical care and guiding treatment pathways, as well as treatment research and clinical audit.

Micro-magnetic resonance imaging study of live quail embryos during embryonic development

Eggs containing live Japanese quail embryos were imaged using micro-magnetic resonance imaging (μMRI) at 24-h intervals from Day 0 to 8, the period during which the main body axis is being laid down and organogenesis is taking place. Considerable detail of non-embryonic structures such as the latebra was revealed at early stages but the embryo could only be visualized around Day 3. Three-dimensional (3D) changes in embryo length and volume were quantified and also changes in volume in the extra- and non-embryonic components. The embryo increased in length by 43% and nearly trebled in volume between Day 4 and Day 5. Although the amount of yolk remained fairly constant over the first 5 days, the amount of albumen decreases significantly and was replaced by extra-embryonic fluid (EEF). 1H longitudinal (T1) and transverse (T2) relaxation times of different regions within the eggs were determined over the first 6 days of development. The T2 measurements mirrored the changes in image intensity observed, which can be related to the aqueous protein concentrations. In addition, a comparison of the development of Day 0 to 3 quail embryos exposed to radiofrequency (rf) pulses, 7 T static magnetic fields and magnetic field gradients for an average of 7 h with the development of control embryos did not reveal any gross changes, thus confirming that μMRI is a suitable tool for following the development of live avian embryos over time from the earliest stages.

Micro-magnetic resonance imaging and embryological analysis of wild-type and pma mutant mice with clubfoot

Gross similarities between the external appearance of the hind limbs of the peroneal muscle atrophy (pma) mouse mutant and congenital talipes equinovarus (CTEV), a human disorder historically referred to as ‘clubfoot’, suggested that this mutant could be a useful model. We used micro‐magnetic resonance imaging to visualize the detailed anatomy of the hind limb defect in mutant pma mice and performed 3D comparisons between mutant and wild‐type hind limbs. We found that the pma foot demonstrates supination (i.e. adduction and inversion of the mid foot and fore foot together with plantar flexion of the ankle and toes) and that the tibiale and distal tarsals display 3D abnormalities in positioning. The size and shape of the tibia, fibula, tarsal and metatarsal bones are similar to the wild‐type. Hypoplasia of the muscles in the antero‐lateral (peroneal) compartment was also demonstrated. The resemblance of these features to those seen in CTEV suggests that the pma mouse is a possibly useful model for the human condition. To understand how the observed deformities in the pma mouse hind foot arise during embryonic development, we followed the process of foot rotation in both wild‐type and pma mutant mice. Rotation of the hind foot in mouse embryos of wild‐type strains (CD‐1 and C57/Black) occurs from embryonic day 14.5 onwards with rotation in C57/Black taking longer. In embryos from both strains, rotation of the right hind foot more commonly precedes rotation of the left. In pma mutants, the initiation of rotation is often delayed and rotation is slower and does not reach completion. If the usefulness of the pma mutant as a model is confirmed, then these findings on pma mouse embryos, when extrapolated to humans, would support a long‐standing hypothesis that CTEV is due to the failure of completion of the normal process of rotation and angulation, historically known as the ‘arrested development hypothesis’.

Decreased adipogenesis and adipose tissue in mice with inactivated protein phosphatase 5

Glucocorticoids play an important role in the treatment of inflammation and immune disorders, despite side effects, which include metabolic derangements such as central adiposity. These studies examine the role of protein phosphatase 5 (Ppp5) in glucocorticoid receptor (GR) complexes which mediate response to glucocorticoids. Mice homozygous for inactivated Ppp5 (Ppp5D274A/D274A) exhibit decreased adipose tissue surrounding the gonads and kidneys compared with wild-type mice. Adipocyte size is smaller, more preadipocytes/stromal cell are present in their gonadal fat tissue and differentiation of preadipocytes to adipocytes is retarded. Glucocorticoid levels are raised and the GR is hyperphosphorylated in adipose tissue of Ppp5D274A/D274A mice at Ser212 and Ser220 (orthologous to human Ser203 and Ser211) in the absence of glucocorticoids. Preadipocyte cultures from Ppp5D274A/D274A mice show decreased down regulation of Delta-like protein-1/preadipocyte factor-1, hyperphosphorylation of extra-cellular signal regulated kinase 2 (ERK2) and increased concentration of (sex determining region Y)-box 9 (SOX9), changes in a pathway essential for preadipocyte differentiation, which leads to decreased concentrations of the transcription factors CEBPβ and CEBPα necessary for the later stages of adipogenesis. The data indicate that Ppp5 plays a crucial role in modifying GR-mediated initiation of adipose tissue differentiation, suggesting that inhibition of Ppp5 may potentially be beneficial to prevent obesity during glucocorticoid treatment.

The developmental and genetic basis of ‘clubfoot’ in the peroneal muscular atrophy mutant mouse

ABSTRACT Genetic factors underlying the human limb abnormality congenital talipes equinovarus (‘clubfoot’) remain incompletely understood. The spontaneous autosomal recessive mouse ‘peroneal muscular atrophy’ mutant (PMA) is a faithful morphological model of human clubfoot. In PMA mice, the dorsal (peroneal) branches of the sciatic nerves are absent. In this study, the primary developmental defect was identified as a reduced growth of sciatic nerve lateral motor column (LMC) neurons leading to failure to project to dorsal (peroneal) lower limb muscle blocks. The pma mutation was mapped and a candidate gene encoding LIM-domain kinase 1 (Limk1) identified, which is upregulated in mutant lateral LMC motor neurons. Genetic and molecular analyses showed that the mutation acts in the EphA4–Limk1–Cfl1/cofilin–actin pathway to modulate growth cone extension/collapse. In the chicken, both experimental upregulation of Limk1 by electroporation and pharmacological inhibition of actin turnover led to defects in hindlimb spinal motor neuron growth and pathfinding, and mimicked the clubfoot phenotype. The data support a neuromuscular aetiology for clubfoot and provide a mechanistic framework to understand clubfoot in humans. Highlighted Article: The mutation in the PMA mouse model of human clubfoot was mapped and a candidate gene, Limk1, identified that was shown to cause sciatic nerve and limb abnormalities when overexpressed.