Performance evaluation of a PET insert for preclinical MRI in stand-alone PET and simultaneous PET-MRI modes.

BACKGROUND: This study aimed to evaluate the performance of a preclinical PET insert in three configurations: as a stand-alone unit outside the MRI bore, inside the bore of a cryogen-free 3T MRI and, finally, while performing simultaneous PET/MRI studies. METHODS: The PET insert consists of two rings of six detectors, each detector comprising 8 × 12 SiPMs reading out dual offset layers of pixelated LYSO crystals with a 1.4-mm pitch. The inner diameter is 60 mm, transaxial field of view (FoV) 40 mm and axial FoV 98 mm. Evaluation was based on NEMA NU 4-2008 guidelines with appropriate modifications. Spatial resolution and sensitivity were measured inside and outside the MR bore. Image quality, count rate and quantitative performance were measured in all three configurations. The effect of temperature stability on PET sensitivity during fast spin echo sequences was also evaluated. B0 field homogeneity and T1 and T2 relaxation times were measured using a water-filled phantom, with and without simultaneous PET operation. Finally, PET and MRI scans of a mouse injected with 10 MBq [18F]NaF and a mouse injected with 16 MBq [18F]FDG were performed in sequential and simultaneous modes. RESULTS: Peak absolute sensitivity was 10.15% with an energy window of 250-750 keV. Absolute sensitivity values outside and inside the MR bore with MR idle agreed to within 0.1%. Outside the MR bore, spatial resolution was 1.21/1.59 mm FWHM (radial/tangential) 5 mm from the centre of the FoV which compared well with 1.19/1.26 mm FWHM inside the MR bore. There were no substantial differences between all three scan configurations in terms of peak NEC rate (175 kcps at 17 MBq), scatter or random fractions. Uniformity and recovery coefficients were also consistent between scanning modes. B0 field homogeneity and T1 and T2 relaxation times were unaltered by the presence of the PET insert. No significant differences were observed between sequential and simultaneous scans of the animals. CONCLUSIONS: We conclude that the performance of the PET insert and MRI system is not significantly affected by the scanning mode.

Collecting duct-derived cells display mesenchymal stem cell properties and retain selective in vitro and in vivo epithelial capacity.

We previously described a mesenchymal stem cell (MSC)-like population within the adult mouse kidney that displays long-term colony-forming efficiency, clonogenicity, immunosuppression, and panmesodermal potential. Although phenotypically similar to bone marrow (BM)-MSCs, kidney MSC-like cells display a distinct expression profile. FACS sorting from Hoxb7/enhanced green fluorescent protein (GFP) mice identified the collecting duct as a source of kidney MSC-like cells, with these cells undergoing an epithelial-to-mesenchymal transition to form clonogenic, long-term, self-renewing MSC-like cells. Notably, after extensive passage, kidney MSC-like cells selectively integrated into the aquaporin 2-positive medullary collecting duct when microinjected into the kidneys of neonatal mice. No epithelial integration was observed after injection of BM-MSCs. Indeed, kidney MSC-like cells retained a capacity to form epithelial structures in vitro and in vivo, and conditioned media from these cells supported epithelial repair in vitro. To investigate the origin of kidney MSC-like cells, we further examined Hoxb7(+) fractions within the kidney across postnatal development, identifying a neonatal interstitial GFP(lo) (Hoxb7(lo)) population displaying an expression profile intermediate between epithelium and interstitium. Temporal analyses with Wnt4(GCE/+):R26(tdTomato/+) mice revealed evidence for the intercalation of a Wnt4-expressing interstitial population into the neonatal collecting duct, suggesting that such intercalation may represent a normal developmental mechanism giving rise to a distinct collecting duct subpopulation. These results extend previous observations of papillary stem cell activity and collecting duct plasticity and imply a role for such cells in collecting duct formation and, possibly, repair.

Absence of neurotoxicity with perineural injection of ultrasound gels: assessment using an animal model.

BACKGROUND: Ultrasound gels may contain propylene glycol and glycerol, which are neurotoxic in high concentrations. If the needle passes through gel during regional anesthesia, gel may be injected near the nerve. It is unknown if this practice poses a risk for neurotoxicity. Using an animal model, we assessed the histological changes of perineural propylene glycol on nerves. We then assessed three commonly used sterile gels for evidence of neurotoxicity. METHODS: Micro-ultrasound guided perineural sciatic nerve injections were performed in mice. Propylene glycol (PG) 2.5%, 10%, 35%, 70% (v/v) or saline was injected. Nerves were assessed after three days for evidence of neurotoxicity. Aquasonic® 100 Ultrasound Gel, K-Y® Lubricating Jelly, and PDI® Lubricating Jelly were also studied against saline controls. RESULTS: Confluent areas of axonal degeneration and intraneural inflammation occurred in 5 of 9 specimens injected with 70% PG. At 35%, 2 of 8 specimens showed patchy changes not present at lower concentrations. No degeneration occurred with Aquasonic® 100 or PDI® Lubricating Jelly. In the K-Y® group, one gel and one saline specimen demonstrated confluent degenerative changes. CONCLUSIONS: Similar to glycerol, 70% PG may cause confluent areas of axon and myelin degeneration with associated intraneural inflammation. The concentration of PG present in ultrasound gels is unlikely to cause neurotoxicity. Aquasonic® 100 and PDI® Lubricating Jelly did not cause neurotoxicity. The results for K-Y® Lubricating Jelly are inconclusive. There is no evidence that passing the needle through the studied gels during regional anesthesia procedures is harmful.

Neuregulin-1 potentiates agrin-induced acetylcholine receptor clustering through muscle-specific kinase phosphorylation.

At neuromuscular synapses, neural agrin (n-agrin) stabilizes embryonic postsynaptic acetylcholine receptor (AChR) clusters by signalling through the muscle-specific kinase (MuSK) complex. Live imaging of cultured myotubes showed that the formation and disassembly of primitive AChR clusters is a dynamic and reversible process favoured by n-agrin, and possibly other synaptic signals. Neuregulin-1 is a growth factor that can act through muscle ErbB receptor kinases to enhance synaptic gene transcription. Recent studies suggest that neuregulin-1-ErbB signalling can modulate n-agrin-induced AChR clustering independently of its effects on transcription. Here we report that neuregulin-1 increased the size of developing AChR clusters when injected into muscles of embryonic mice. We investigated this phenomenon using cultured myotubes, and found that in the ongoing presence of n-agrin, neuregulin-1 potentiates AChR clustering by increasing the tyrosine phosphorylation of MuSK. This potentiation could be blocked by inhibiting Shp2, a postsynaptic tyrosine phosphatase known to modulate the activity of MuSK. Our results provide new evidence that neuregulin-1 modulates the signaling activity of MuSK and hence might function as a second-order regulator of postsynaptic AChR clustering at the neuromuscular synapse. Thus two classic synaptic signalling systems (neuregulin-1 and n-agrin) converge upon MuSK to regulate postsynaptic differentiation.

Multiple non-cell-autonomous defects underlie neocortical callosal dysgenesis in Nfib-deficient mice.

BACKGROUND: Agenesis of the corpus callosum is associated with many human developmental syndromes. Key mechanisms regulating callosal formation include the guidance of axons arising from pioneering neurons in the cingulate cortex and the development of cortical midline glial populations, but their molecular regulation remains poorly characterised. Recent data have shown that mice lacking the transcription factor Nfib exhibit callosal agenesis, yet neocortical callosal neurons express only low levels of Nfib. Therefore, we investigate here how Nfib functions to regulate non-cell-autonomous mechanisms of callosal formation. RESULTS: Our investigations confirmed a reduction in glial cells at the midline in Nfib-/- mice. To determine how this occurs, we examined radial progenitors at the cortical midline and found that they were specified correctly in Nfib mutant mice, but did not differentiate into mature glia. Cellular proliferation and apoptosis occurred normally at the midline of Nfib mutant mice, indicating that the decrease in midline glia observed was due to deficits in differentiation rather than proliferation or apoptosis. Next we investigated the development of callosal pioneering axons in Nfib-/- mice. Using retrograde tracer labelling, we found that Nfib is expressed in cingulate neurons and hence may regulate their development. In Nfib-/- mice, neuropilin 1-positive axons fail to cross the midline and expression of neuropilin 1 is diminished. Tract tracing and immunohistochemistry further revealed that, in late gestation, a minor population of neocortical axons does cross the midline in Nfib mutants on a C57Bl/6J background, forming a rudimentary corpus callosum. Finally, the development of other forebrain commissures in Nfib-deficient mice is also aberrant. CONCLUSION: The formation of the corpus callosum is severely delayed in the absence of Nfib, despite Nfib not being highly expressed in neocortical callosal neurons. Our results indicate that in addition to regulating the development of midline glial populations, Nfib also regulates the expression of neuropilin 1 within the cingulate cortex. Collectively, these data indicate that defects in midline glia and cingulate cortex neurons are associated with the callosal dysgenesis seen in Nfib-deficient mice, and provide insight into how the development of these cellular populations is controlled at a molecular level.

Developmental regulation of cardiovascular function is dependent on both genotype and environment.

Adverse developmental environments can increase the risk of adult cardiovascular disease, but not all individuals are affected, suggesting the importance of genotype. Genetically distinct mouse strains allow the genetic dissection of complex traits; however, they have not been used to evaluate the developmental origins of adult cardiovascular disease. Our objective was to determine the effect of prenatal nutrient restriction (R) on adult cardiovascular function in A/J (AJ) and C57BL/6J (B6) mice and whether a postnatal high-fat (HF) diet exacerbates these effects. Pregnant AJ and B6 mice underwent a 30% R or ad libitum diet, and their offspring underwent a HF or control diet. Hypertension (+17 mmHg; P<0.001) was observed in B6R mice at 9 wk, and their arterial pressure tended to remain high at 25 wk (+13 mmHg; not significant). In AJR mice, the normal decrement in arterial pressure over this age range in this strain was abolished. Heart rate prematurely increased in B6R and decreased in AJR (all; P<0.05) mice from 9 to 25 wk. There was no effect of postnatal HF diet on these relationships. The Tei index (from a 26-wk microultrasound) was increased in both AJR and B6R mice (all; P<0.05), suggesting an improved global myocardial performance. Neither R nor HF alone changed diastolic (ratio of E wave to A wave) or systolic (%fractional shortening) function in either strain; however, R and HE combined improved diastolic function in B6 (P<0.05) but not in AJ mice. Therefore, there are strain-dependent alterations in adult cardiovascular function in response to prenatal nutrient restriction. Unexpectedly, a postnatal HF diet did not exacerbate the effects of prenatal nutrient restriction on postnatal cardiovascular outcomes.

Nuclear factor I gene expression in the developing forebrain.

Three members of the Nuclear Factor I (Nfi) gene family of transcription factors; Nfia, Nfib, and Nfix are highly expressed in the developing mouse brain. Nfia and Nfib knockout mice display profound defects in the development of midline glial populations and the development of forebrain commissures (das Neves et al. [1999] Proc Natl Acad Sci U S A 96:11946-11951; Shu et al. [2003] J Neurosci 23:203-212; Steele-Perkins et al. [2005] Mol Cell Biol 25:685-698). These findings suggest that Nfi genes may regulate the substrate over which the commissural axons grow to reach targets in the contralateral hemisphere. However, these genes are also expressed in the cerebral cortex and, thus, it is important to assess whether this expression correlates with a cell-autonomous role in cortical development. Here we detail the protein expression of NFIA and NFIB during embryonic and postnatal mouse forebrain development. We find that both NFIA and NFIB are expressed in the deep cortical layers and subplate prenatally and display dynamic expression patterns postnatally. Both genes are also highly expressed in the developing hippocampus and in the diencephalon. We also find that principally neither NFIA nor NFIB are expressed in callosally projecting neurons postnatally, emphasizing the role for midline glial cell populations in commissure formation. However, a large proportion of laterally projecting neurons express both NFIA and NFIB, indicating a possible cell-autonomous role for these transcription factors in corticospinal neuron development. Collectively, these data suggest that, in addition to regulating the formation of axon guidance substrates, these genes also have cell-autonomous roles in cortical development.

Embryonic and neonatal phenotyping of genetically engineered mice.

Considerable progress has been made in adapting existing and developing new technologies to enable increasingly detailed phenotypic information to be obtained in embryonic and newborn mice. Sophisticated methods for imaging mouse embryos and newborns are available and include ultrasound and magnetic resonance imaging (MRI) for in vivo imaging, and MRI, vascular corrosion casts, micro-computed tomography, and optical projection tomography (OPT) for postmortem imaging. In addition, Doppler and M-mode ultrasound are useful noninvasive tools to monitor cardiac and vascular hemodynamics in vivo in embryos and newborns. The developmental stage of the animals being phenotyped is an important consideration when selecting the appropriate technique for anesthesia or euthanasia and for labeling animals in longitudinal studies. Study design also needs to control for possible differences between inter- and intralitter variability, and for possible long-term developmental effects caused by anesthesia and/or procedures. Noninvasive or minimally invasive intravenous or intracardiac injections or blood sampling, and arterial pressure and electrocardiography (ECG) measurements are feasible in newborns. Whereas microinjection techniques are available for embryos as young as 6.5 days of gestation, further advances are required to enable minimally invasive fluid or tissue samples, or blood pressure or ECG measurements, to be obtained from mouse embryos in utero. The growing repertoire of techniques available for phenotyping mouse embryos and newborns promises to accelerate knowledge gained from studies using genetically engineered mice to understand molecular regulation of morphogenesis and the etiology of congenital diseases.

High resolution ultrasound-guided microinjection for interventional studies of early embryonic and placental development in vivo in mice.

BACKGROUND: In utero microinjection has proven valuable for exploring the developmental consequences of altering gene expression, and for studying cell lineage or migration during the latter half of embryonic mouse development (from embryonic day 9.5 of gestation (E9.5)). In the current study, we use ultrasound guidance to accurately target microinjections in the conceptus at E6.5-E7.5, which is prior to cardiovascular or placental dependence. This method may be useful for determining the developmental effects of targeted genetic or cellular interventions at critical stages of placentation, gastrulation, axis formation, and neural tube closure. RESULTS: In 40 MHz ultrasound images at E6.5, the ectoplacental cone region and proamniotic cavity could be visualized. The ectoplacental cone region was successfully targeted with 13.8 nL of a fluorescent bead suspension with few or no beads off-target in 51% of concepti microinjected at E6.5 (28/55 injected). Seventy eight percent of the embryos survived 2 to 12 days post injection (93/119), 73% (41/56) survived to term of which 68% (38/56) survived and appeared normal one week after birth. At E7.5, the amniotic and exocoelomic cavities, and ectoplacental cone region were discernable. Our success at targeting with few or no beads off-target was 90% (36/40) for the ectoplacental cone region and 81% (35/43) for the exocoelomic cavity but tended to be less, 68% (34/50), for the smaller amniotic cavity. At E11.5, beads microinjected at E7.5 into the ectoplacental cone region were found in the placental spongiotrophoblast layer, those injected into the exocoelomic cavity were found on the surface or within the placental labyrinth, and those injected into the amniotic cavity were found on the surface or within the embryo. Following microinjection at E7.5, survival one week after birth was 60% (26/43) when the amniotic cavity was the target and 66% (19/29) when the target was the ectoplacental cone region. The survival rate was similar in sham experiments, 54% (33/61), for which procedures were identical but no microinjection was performed, suggesting that surgery and manipulation of the uterus were the main causes of embryonic death. CONCLUSION: Ultrasound-guided microinjection into the ectoplacental cone region at E6.5 or E7.5 and the amniotic cavity at E7.5 was achieved with a 7 day postnatal survival of >/=60%. Target accuracy of these sites and of the exocoelomic cavity at E7.5 was > or =51%. We suggest that this approach may be useful for exploring gene function during early placental and embryonic development.