Longitudinal in Utero Analysis of Engrailed-1 Knockout Mouse Embryonic Phenotypes Using High-Frequency Ultrasound

Large-scale international efforts to generate and analyze loss-of-function mutations in each of the approximately 20,000 protein-encoding gene mutations are ongoing using the “knockout” mouse as a model organism. Because one-third of gene knockouts are expected to result in embryonic lethality, it is important to develop non-invasive in utero imaging methods to detect and monitor mutant phenotypes in mouse embryos. We describe the utility of 3-D high-frequency (40-MHz) ultrasound (HFU) for longitudinal in utero imaging of mouse embryos between embryonic days (E) 11.5 and E14.5, which represent critical stages of brain and organ development. Engrailed-1 knockout (En1-ko) mouse embryos and their normal control littermates were imaged with HFU in 3-D, enabling visualization of morphological phenotypes in the developing brains, limbs and heads of the En1-ko embryos. Recently developed deep learning approaches were used to automatically segment the embryonic brain ventricles and bodies from the 3-D HFU images, allowing quantitative volumetric analyses of the En1-ko brain phenotypes. Taken together, these results show great promise for the application of longitudinal 3-D HFU to analyze knockout mouse embryos in utero.     

THE ADAPTATION OF UNMODIFIED STRAINS OF YELLOW FEVER VIRUS TO CULTIVATION IN VITRO

1. In a search for suitable tissues for the cultivation of yellow fever virus in vitro, mouse embryos were inoculated with this virus in utero. A titration for virus content of the various organs of the embryos indicated that the virus was present in the brain in greatest concentration. 2. Unmodified strains of yellow fever virus were readily adapted to cultivation in vitro in a medium consisting of minced mouse embryo brain tissue and Tyrode solution containing 10 per cent normal monkey serum. 3. After a continued cultivation in mouse embryo brain tissue cultures for twenty to twenty-five subcultures, these strains were readily adapted to cultivation in whole mouse embryo tissue medium. 4. There is evidence to indicate that a prolonged cultivation of the virus in mouse embryo brain medium increases its neurotropic properties. 5. Attempts to employ monkey tissues for in vitro cultivation of yellow fever virus gave entirely negative results.     

High-Frequency Chirp Ultrasound Imaging with an Annular Array for Ophthalmologic and Small-Animal Imaging

High-frequency ultrasound (HFU, >20 MHz) is an attractive means of obtaining fine-resolution images of biological tissues for ophthalmologic, dermatological and small-animal imaging applications. Even with current improvements in circuit designs and high-frequency equipment, HFU has two inherent limitations. First, HFU images have a limited depth-of-field (DOF) because of the short wavelength and the low fixed F-number of conventional HFU transducers. Second, HFU is usually limited to shallow imaging because of the significant attenuation in most tissues. In a previous study, a five-element annular array with a 17-MHz center frequency was excited using chirp-coded signals, and a synthetic-focusing algorithm was used to extend the DOF and increase penetration depth. In the present study, a similar approach with two different five-element annular arrays operating near a center frequency of 35 MHz is implemented and validated. Following validation studies, the chirp-imaging methods were applied to imaging vitreous-hemorrhage-mimicking phantoms and mouse embryos. Images of the vitreous phantom showed increased sensitivity using the chirp method compared with a standard monocycle imaging method, and blood droplets could be visualized 4 mm deeper into the phantom. Three-dimensional datasets of 12.5-day-old mouse embryo heads were acquired in utero using chirp and conventional excitations. Images were formed and brain ventricles were segmented and reconstructed in three dimensions. The brain ventricle volumes for the monocycle excitation exhibited artifacts that were not apparent on the chirp-based dataset reconstruction. (E-mail: mamou@rrinyc.org)     

VEGFR2-Targeted Molecular Imaging in the Mouse Embryo: An Alternative to the Tumor Model

As a tumor surrogate, the mouse embryo presents as an excellent alternative for examining the binding of angiogenesis-targeting microbubbles and assessing the quantitative nature of molecular ultrasound. We establish the validity of this model by developing a robust method to study microbubble kinetic behavior and investigate the reproducibility of targeted binding in the murine embryo. Vascular endothelial growth factor receptor 2 (VEGFR2)-targeted (MB_V), rat immunoglobulin G₂ (IgG₂) control antibody-targeted (MB_C) and untargeted (MB_U) microbubbles were introduced into vasculature of living mouse embryos. Non-linear contrast-specific and B-mode ultrasound imaging, performed at 21 MHz with a Vevo-2100 scanner, was used to collect basic perfusion parameters and contrast mean power ratios for all bubble types. We observed a twofold increase (p < 0.001) in contrast mean power ratios for MB_V (4.14 ± 1.78) compared with those for MB_C (1.95 ± 0.78) and MB_U (1.79 ± 0.45). Targeted imaging of endogenous endothelial cell surface markers in mouse embryos is possible with labeled microbubbles. The mouse embryo thus presents as a versatile model for testing the performance of ultrasound molecular targeting, where further development of quantitative imaging techniques may enable rapid evaluations of biomarker expression in studies of vascular development, disease and angiogenesis.     

Acrocephalosyndactyly,Apert type,in a newborn: Cerebral sonography

We describe the clinical and cerebral ultrasonographic features of a rare case of type 1 acrocephalosyndactyly (Apert syndrome). The patient was a newborn male whose twin had died in utero. Most cases of Apert syndrome are sporadic, although autosomal dominant inheritance has also been reported. Diagnosis is based on physical examination together with imaging data. Since Apert syndrome can give rise to numerous CNS abnormalities, affected newborns should undergo echoencephalography for more complete characterization of their malformations.     

Measurement of cerebral blood volume in mouse brain regions using micro-computed tomography

Micro-computed tomography (micro-CT) is an X-ray imaging technique that can produce detailed 3D images of cerebral vasculature. This paper describes the development of a novel method for using micro-CT to measure cerebral blood volume (CBV) in the mouse brain. As an application of the methodology, we test the hypotheses that differences in CBV exist over anatomical brain regions and that high energy demanding primary sensory regions of the cortex have locally elevated CBV, which may reflect a vascular specialization. CBV was measured as the percentage of tissue space occupied by a radio-opaque silicon rubber that fills the vasculature. To ensure accuracy of the CBV measurements, several innovative refinements were made to standard micro-CT specimen preparation and analysis procedures. Key features of the described method are vascular perfusion under controlled pressure, registration of the micro-CT images to an MRI anatomical brain atlas and re-scaling of micro-CT intensities to CBV units with selectable exclusion of major vessels. Histological validation of the vascular perfusion showed that the average percentage of vessels filled was 93 ± 3%. Comparison of thirteen brain regions in nine mice revealed significant differences in CBV between regions (p  < 0.0001) while cortical maps showed that primary visual and auditory areas have higher CBV than primary somatosensory areas.     

Establishing an Animal Model of Intracerebral Hemorrhage under the Guidance of Ultrasound

We established an intracerebral hemorrhage (ICH) model by puncturing the middle cerebral artery (MCA) under ultrasonic guidance and used the same model to evaluate the efficiency of ICH detection with contrast-enhanced ultrasound (CEUS). MCAs of 12 adult dogs were punctured to bleed under ultrasonic guidance. CEUS of the brain was performed every 30 min until a hematoma formed. A subsequent CEUS was performed after 24 h, after which brain samples were collected for pathologic examination. Eleven of the 12 dogs successfully developed ICH as confirmed by CEUS and pathologic examination. The hematoma diameters as determined through CEUS and pathologic examination were 22.4 mm (SD = 7.1) and 21.6 mm (SD = 6.9), respectively. No significant difference was found between the measurements by the two methods (p > 0.05). The CEUS results indicated characteristics of active bleeding and hematoma. Hence, a dog ICH model was established by puncturing the MCA under ultrasonic guidance, and imaging characteristics were in good accordance with those of patients.     

Percutaneous left ventricular assist device can prevent acute cerebral ischaemia during ventricular fibrillation

AimsA percutaneous left ventricular assist device has been shown to be able to perfuse cardiac and cerebral tissues during cardiac arrest and may be a useful supplement to current methods in resuscitation. We wished to assess device-assisted circulation during cardiac arrest with microspheres injections and continuous end-tidal CO₂ monitoring, and used cerebral microdialysis to detect ischaemia in the brain.Methods12 anaesthetised pigs had microdialysis and pressure catheters implanted via craniotomy. The percutaneous assist device was deployed transfemorally. Ventricular fibrillation was induced by angioplasty-balloon occlusion of the left coronary artery. Cerebral microdialysis samples representing 0–20 and 20–40 min of cardiac arrest with assisted circulation were analysed for markers of cerebral injury (glucose, pyruvate, lactate, and glycerol).ResultsMicrodialysis showed no ischaemic changes after 20 min of cardiac arrest (P = NS to Baseline for glucose, glycerol, lactate, pyruvate and lactate/pyruvate ratio) in subjects with maintained end-tidal CO₂ values above 1.3 kPa (10 mmHg). After 40 min only lactate showed a significant change compared to Baseline (P < 0.05). Microspheres flow to the brain was 57% and myocardial flow was 72% compared to Baseline after 15 min (P < 0.05). After 45 min flow declined to 22% and 40% of Baseline, respectively (P = NS vs. 15 min).ConclusionsA percutaneous left ventricular assist device may prevent ischaemic cerebral injury during cardiac arrest for a limited time. Cerebral injury and tissue perfusion were indicated by end-tidal CO₂.     

Efficacy of diffusion-weighted magnetic resonance imaging performed before therapeutic hypothermia in predicting clinical outcome in comatose cardiopulmonary arrest survivors

Aim of the studyTo develop a clinically relevant and qualitative brain magnetic resonance imaging (MRI) scoring system for acute stage comatose cardiac arrest patients.MethodsConsecutive comatose post-cardiopulmonary arrest patients were prospectively enrolled. Routine brain MRI sequences were scored by two independent and blinded experts. Predefined brain regions were qualitatively scored on diffusion-weighted imaging (DWI) sequences according to the severity of the abnormality on a scale from 0 to 4. The mean score provided by the raters determined poor outcome defined under the Cerebral Performance Categories 3, 4, or 5. DWI scans were repeated after therapeutic hypothermia (TH). The same qualitative scoring system was applied and results were compared to the initial scores.ResultsOut of 24 recruited patients, 19 with brain MRI scans were included. Of the 19 included patients, seven showed a good outcome at hospital discharge and 12 patients showed poor neurologic outcome. Median time from the arrest to the initial DWI was 166 min (IQR 114–240 min). At 100% specificity, the overall, cortex, and cortex plus deep grey nuclei scores predicted poor patient outcome with a sensitivity of 91.7–100% (95% CI). Follow-up DWI scans after TH showed worse results than initial scans.ConclusionA qualitative MRI scoring system effectively assessed the severity of hypoxic-ischaemic brain injury following cardiopulmonary arrest. The scoring system may provide useful prognostic information in comatose cardiopulmonary arrest patients.     

Spectroscopic second and third harmonic generation microscopy using a femtosecond laser source in the third near-infrared (NIR-III) optical window

In this study, second harmonic generation (SHG) and third harmonic generation (THG) spectroscopic imaging were performed on biological samples using a femtosecond laser source in the third near-infrared (NIR) optical window (NIR-III). Using a visible-NIR spectrometer, the SHG and THG signals were simultaneously detected and were extracted using spectral analysis. Visualization of biological samples such as cultured cells (HEK293 T), mouse brain slices, and the nematode Caenorhabditis elegans was performed in a label-free manner. In particular, in an SHG image of an entire coronal brain section (8 × 6 mm²), we observed mesh-like and filamentous structures in the arachnoid mater and wall of the cerebral ventricle, probably corresponding to the collagen fibers, cilia, and rootlet. Moreover, the THG images clearly depicted the densely packed axons in the white matter and cell nuclei at the cortex of the mouse brain slice sample and lipid-rich granules such as lipid droplets inside the nematode. The observations and conclusions drawn from this technique confirm that it can be utilized for various biological applications, including in vivo label-free imaging of living animals.     

Fast quantitative mapping of absolute water content with full brain coverage

Quantitative mapping of water content, especially in the human brain, has the potential to provide important information for the study and diagnosis of diseases associated with a focal or global change in tissue water homeostasis. In the current work, an imaging method for the precise and accurate quantification of tissue water content is presented. The method allows the acquisition of water content maps with voxel dimensions of 1 × 1 × 2 mm³ and full brain coverage in less than 10 min on a standard clinical 1.5 T scanner. The precision was optimised for human brain imaging and possible sources of systematic error were carefully investigated, demonstrating the ability of the method to quantify water content with high accuracy and precision. The approach was validated in phantom experiments and quantitative cerebral water content maps of a group of 10 healthy volunteers were obtained.     

A Method to Validate Quantitative High-Frequency Power Doppler Ultrasound With Fluorescence in Vivo Video Microscopy

Flow quantification with high-frequency (>20 MHz) power Doppler ultrasound can be performed objectively using the wall-filter selection curve (WFSC) method to select the cutoff velocity that yields a best-estimate color pixel density (CPD). An in vivo video microscopy system (IVVM) is combined with high-frequency power Doppler ultrasound to provide a method for validation of CPD measurements based on WFSCs in mouse testicular vessels. The ultrasound and IVVM systems are instrumented so that the mouse remains on the same imaging platform when switching between the two modalities. In vivo video microscopy provides gold-standard measurements of vascular diameter to validate power Doppler CPD estimates. Measurements in four image planes from three mice exhibit wide variation in the optimal cutoff velocity and indicate that a predetermined cutoff velocity setting can introduce significant errors in studies intended to quantify vascularity. Consistent with previously published flow-phantom data, in vivo WFSCs exhibited three characteristic regions and detectable plateaus. Selection of a cutoff velocity at the right end of the plateau yielded a CPD close to the gold-standard vascular volume fraction estimated using IVVM. An investigator can implement the WFSC method to help adapt cutoff velocity to current blood flow conditions and thereby improve the accuracy of power Doppler for quantitative microvascular imaging.     

Clinical,laboratory and brain Magnetic Resonance Imaging (MRI) characteristics of asymptomatic and symptomatic HIV-negative neurosyphilis patients

IntroductionThere are few studies concerning the differences between asymptomatic neurosyphilis (ANS) and symptomatic neurosyphilis (SNS). This study aimed to summarize clinical, laboratory and brain Magnetic Resonance Imaging (MRI) characteristics of HIV-negative patients with ANS and SNS.MethodsData from 43 HIV-negative patients with ANS and 59 HIV-negative patients with SNS were retrospectively collected from our hospital between December 2012 and December 2018.ResultsCompared with the ANS group, SNS group had more patients that were male, age≥45 years, had brain MRI abnormalities, and exhibited higher serum/cerebrospinal fluid (CSF) TRUST titer, CSF WBC count, CSF protein concentration (P < 0.05). Multivariate regression analysis revealed that male sex, age ≥45 years and CSF TRUST titer were risk factors for SNS [odds ratio (OR) = 7.946,P = 0.001;OR = 3.757, P = 0.041; OR = 2.713, P = 0.002; respectively]. The brain MRI findings of 78 patients without comorbidities showed that ischemic infarct lesions presented in 17/37 (45.95%) of patients with ANS; infarct ischemic stroke (73.17%) especially multiple cerebral infractions (46.34%), cerebral atrophy (48.78%) were also common presentations in the SNS group.ConclusionsPatients with HIV-negative ANS and SNS presented different clinical, laboratory and brain MRI features. Male sex, age ≥45 years and elevated CSF TRUST titer may have an increased risk of developing neurological symptoms. Brain MRI abnormalities may present prior to clinical symptoms. Multiple cerebral infarctions without explained reasons or cerebral atrophy should alert clinicians the possibility of SNS.     

R-fMRI reconstruction from k–t undersampled data using a subject-invariant dictionary model and VB-EM with nested minorization

Higher spatial resolution in resting-state functional magnetic resonance imaging (R-fMRI) can give reliable information about the functional networks in the cerebral cortex. Typical methods can achieve higher spatial or temporal resolution by speeding up scans using either (i) complex pulse-sequence designs or (ii) k-space undersampling coupled with priors on the signal. We propose to undersample the R-fMRI acquisition in k-space and time to speedup scans in order to improve spatial resolution. We propose a novel model-based R-fMRI reconstruction framework using a robust, subject-invariant, spatially regularized dictionary prior on the signal. Furthermore, we propose a novel inference framework based on variational Bayesian expectation maximization with nested minorization (VB-EM-NM). Our inference framework allows us to provide an estimate of uncertainty of the reconstruction, unlike typical reconstruction methods. Empirical evaluation of (i) simulated R-fMRI reconstruction and (ii) functional-network estimates from brain R-fMRI reconstructions demonstrate that our framework improves over the state of the art, and, additionally, enables significantly higher spatial resolution.     

Subharmonic,Non-linear Fundamental and Ultraharmonic Imaging of Microbubble Contrast at High Frequencies

There is increasing use of ultrasound contrast agent in high-frequency ultrasound imaging. However, conventional contrast detection methods perform poorly at high frequencies. We performed systematic in vitro comparisons of subharmonic, non-linear fundamental and ultraharmonic imaging for different depths and ultrasound contrast agent concentrations (Vevo 2100 system with MS250 probe and MicroMarker ultrasound contrast agent, VisualSonics, Toronto, ON, Canada). We investigated 4-, 6- and 10-cycle bursts at three power levels with the following pulse sequences: B-mode, amplitude modulation, pulse inversion and combined pulse inversion/amplitude modulation. The contrast-to-tissue (CTR) and contrast-to-artifact (CAR) ratios were calculated. At a depth of 8 mm, subharmonic pulse-inversion imaging performed the best (CTR = 26 dB, CAR = 18 dB) and at 16 mm, non-linear amplitude modulation imaging was the best contrast imaging method (CTR = 10 dB). Ultraharmonic imaging did not result in acceptable CTRs and CARs. The best candidates from the in vitro study were tested in vivo in chicken embryo and mouse models, and the results were in a good agreement with the in vitro findings.     

Intraoperative dynamic susceptibility contrast MRI (iDSC-MRI) is as reliable as preoperatively acquired perfusion mapping

DSC-MRI was applied intraoperatively during human brain tumor removal. Immediately after complete tumor resection was presumed, MRI including a dynamic susceptibility contrast T2?-weighted EPI sequence was performed in 30 patients while the skull was still open using a flexible two-channel coil system at an intraoperative 1.5-Tesla MR scanner. Maps of relative regional blood flow (rCBF), blood volume (rCBV), and mean transit time (MTT) were calculated, and ratios of these maps were compared to preoperatively acquired DSC-MRI data. The extent of the resection was compared with the postoperative MRI performed 24 h after the operation. In 8 of these patients residual tumor tissue was depicted at the time of intraoperative MRI. In corresponding regions ratios for rCBV and rCBF did not differ significantly between pre- and intraoperatively acquired data (two-tailed t-test). Furthermore, we found a high correlation between ratios created from pre- and intraoperatively measured data for both rCBV and rCBF, respectively (Pearson correlation; r²_rCBV = 0.86, p ≤ 0.01; r²_rCBF = 0.86, p ≤ 0.01). DSC-MRI is a powerful tool for the differential diagnosis of brain lesions. Its use intraoperatively provides pathophysiologic information that is up-to-date, independently of an intraoperative brain shift and also independently of the known leakage phenomenon caused by surgical manipulation. It can assist in the decision to depict residual tumor burden beyond conventional imaging. Our data demonstrate that iDSC-MRI is as reliable as preoperatively acquired data.     

Sulfate-sulfur metabolism in the rat fetus as indicated by sulfur-35

Twenty-four hours after the intraperitoneal injection of sodium sulfate-S³⁵ into pregnant rats, sulfur-35 was found in the embryos. The amount of the sulfur-35 retained by the embryos was directly related to their degree of development in utero. A large fraction of the sulfur-35 found in the embryos was insoluble in 5 per cent trichloroacetic acid. At the 9th to 10th day of development, about 40 per cent of the sulfur-35 was present in this fraction. In 20-day-old embryos this fraction accounted for nearly 90 per cent of the total. Radioautographs of sections of embryos fixed in a solution of formaldehyde revealed that the sulfur-35 was most highly concentrated in the cartilaginous portion of the skeleton. All other tissues gave much weaker autographic reactions, comparable with the over-all reaction obtained when sections from embryos fixed in a solution of formaldehyde saturated with barium hydroxide were used. By analysis for the sulfur-35 content of individual tissues the concentration of the sulfur-35 in humeri from 20-day-old embryos was found to be about 30 times that in the maternal sternum. The concentration of the isotope in the skeletal muscle, brain, heart, and skin of the same embryos was also higher than in the corresponding maternal tissues. On the other hand, the concentration of the sulfur-35 in the maternal gastrointestinal tract plus contents was higher than in the gastrointestinal tract and contents of the embryos.     

MR-elastography reveals degradation of tissue integrity in multiple sclerosis

In multiple sclerosis (MS), diffuse brain parenchymal damage exceeding focal inflammation is increasingly recognized to be present from the very onset of the disease, and, although occult to conventional imaging techniques, may present a major cause of permanent neurological disability. Subtle tissue alterations significantly influence biomechanical properties given by stiffness and internal friction, that – in more accessible organs than the brain – are traditionally assessed by manual palpation during the clinical exam. The brain, however, is protected from our sense of touch, and thus our current knowledge on cerebral viscoelasticity is very limited. We developed a clinically feasible magnetic resonance elastography setup sensitive to subtle alterations of brain parenchymal biomechanical properties. Investigating 45 MS patients revealed a significant decrease (13%, P < 0.001) of cerebral viscoelasticity compared to matched healthy volunteers, indicating a widespread tissue integrity degradation, while structure-geometry defining parameters remained unchanged. Cerebral viscoelasticity may represent a novel in vivo marker of neuroinflammatory and neurodegenerative pathology.     

Dual modality optical coherence and whole-body photoacoustic tomography imaging of chick embryos in multiple development stages

Chick embryos are an important animal model for biomedical studies. The visualization of chick embryos, however, is limited mostly to postmortem sectional imaging methods. In this work, we present a dual modality optical imaging system that combines swept-source optical coherence tomography and whole-body photoacoustic tomography, and apply it to image chick embryos at three different development stages. The explanted chick embryos were imaged in toto with complementary contrast from both optical scattering and optical absorption. The results serve as a prelude to the use of the dual modality system in longitudinal whole-body monitoring of chick embryos in ovo.OCIS codes: (170.4500) Optical coherence tomography, (170.5120) Photoacoustic imaging, (170.3880) Medical and biological imaging     

Differences in coordination and timing of pre-reaching upper extremity movements may be an indicator of cerebral palsy in infants with stroke: A preliminary investigation

BackgroundNeonatal stroke is a leading cause of hemiplegic cerebral palsy that occurs around the time of birth. Infants are diagnosed with cerebral palsy when motor impairments become clinically apparent, months or years after the stroke. Tools/methods for identifying high risk or diagnosis of cerebral palsy in infancy are improving.MethodsWe measured spatial and temporal kinematics of pre-reaching upper extremity movements in 2–3 month old infants with neonatal stroke and typical development. We aimed to evaluate the feasibility of applying kinematics in this population and collect preliminary data to explore (1) if asymmetries are present in the infants with neonatal stroke, particularly those with a later diagnosis of cerebral palsy, and (2) to compare differences in the timing and coordination of their movements to infants with typical development, and infants with stroke and no cerebral palsy. Participants were 21 full-term infants, 10 with stroke (4 who later received a cerebral palsy diagnosis) age 72.1 (SD 9.3) days, and 11 typically developing, age 74.3 (SD 9.3) days.FindingsResults showed that infants with stroke and cerebral palsy demonstrated significant asymmetry in the average movement length (p = 0.0089) and hand path length (p = 0.0275) between their involved and uninvolved sides and moved less frequently (p = 0.09) and slower (p = 0.041) than infants with stroke and no cerebral palsy.InterpretationResults suggest that kinematic analysis might detect asymmetries and motor impairment indicative of hemiplegic cerebral palsy earlier than current assessments and that asymmetry in speed, length and frequency of arm movements may be early indicators. This study is preliminary, limiting interpretation of the results.