Automated Computational Processing of 3-D MR Images of Mouse Brain for Phenotyping of Living Animals

Magnetic resonance (MR) imaging provides a method to obtain anatomical information from the brain in vivo that is not typically available by optical imaging because of this organ's opacity. MR is nondestructive and obtains deep tissue contrast with 100-µm^3 voxel resolution or better. Manganese-enhanced MRI (MEMRI) may be used to observe axonal transport and localized neural activity in the living rodent and avian brain. Such enhancement enables researchers to investigate differences in functional circuitry or neuronal activity in images of brains of different animals. Moreover, once MR images of a number of animals are aligned into a single matrix, statistical analysis can be done comparing MR intensities between different multi-animal cohorts comprising individuals from different mouse strains or different transgenic animals, or at different time points after an experimental manipulation. Although preprocessing steps for such comparisons (including skull stripping and alignment) are automated for human imaging, no such automated processing has previously been readily available for mouse or other widely used experimental animals, and most investigators use in-house custom processing. This protocol describes a stepwise method to perform such preprocessing for mouse

Decoupling the Effects of the Amyloid Precursor Protein From Amyloid-β Plaques on Axonal Transport Dynamics in the Living Brain

Amyloid precursor protein (APP) is the precursor to Aβ plaques. The cytoplasmic domain of APP mediates attachment of vesicles to molecular motors for axonal transport. In APP-KO mice, transport of Mn²⁺ is decreased. In old transgenic mice expressing mutated human (APP^(SwInd)) linked to Familial Alzheimer’s Disease, with both expression of APP^(SwInd) and plaques, the rate and destination of Mn²⁺ axonal transport is altered, as detected by time-lapse manganese-enhanced magnetic resonance imaging (MEMRI) of the brain in living mice. To determine the relative contribution of expression of APP^(SwInd) versus plaque on transport dynamics, we developed a Tet-off system to decouple expression of APP^(SwInd) from plaque, and then studied hippocampal to forebrain transport by MEMRI. Three groups of mice were compared to wild-type (WT): Mice with plaque and APP^(SwInd) expression; mice with plaque but suppression of APP^(SwInd) expression; and mice with APP^(SwInd) suppressed from mating until 2 weeks before imaging with no plaque. MR images were captured before at successive time points after stereotactic injection of Mn²⁺ (3–5 nL) into CA3 of the hippocampus. Mice were returned to their home cage between imaging sessions so that transport would occur in the awake freely moving animal. Images of multiple mice from the three groups (suppressed or expressed) together with C57/B6J WT were aligned and processed with our automated computational pipeline, and voxel-wise statistical parametric mapping (SPM) performed. At the conclusion of MR imaging, brains were harvested for biochemistry or histopathology. Paired T-tests within-group between time points (p = 0.01 FDR corrected) support the impression that both plaque alone and APP^(SwInd) expression alone alter transport rates and destination of Mn²⁺ accumulation. Expression of APP^(SwInd) in the absence of plaque or detectable Aβ also resulted in transport defects as well as pathology of hippocampus and medial septum, suggesting two sources of pathology occur in familial Alzheimer’s disease, from toxic mutant protein as well as plaque. Alternatively mice with plaque without APP^(SwInd) expression resemble the human condition of sporadic Alzheimer’s, and had better transport. Thus, these mice with APP^(SwInd) expression suppressed after plaque formation will be most useful in preclinical trials

A simple rapid process for semi-automated brain extraction from magnetic resonance images of the whole mouse head

Background: Magnetic resonance imaging (MRI) is a well-developed technique in neuroscience. Limitations in applying MRI to rodent models of neuropsychiatric disorders include the large number of animals required to achieve statistical significance, and the paucity of automation tools for the critical early step in processing, brain extraction, which prepares brain images for alignment and voxel-wise statistics. New Method: This novel timesaving automation of template-based brain extraction (“skull-stripping”) is capable of quickly and reliably extracting the brain from large numbers of whole head images in a single step. The method is simple to install and requires minimal user interaction. Results: This method is equally applicable to different types of MR images. Results were evaluated with Dice and Jacquard similarity indices and compared in 3D surface projections with other stripping approaches. Statistical comparisons demonstrate that individual variation of brain volumes are preserved. Comparison with Existing Methods: A downloadable software package not otherwise available for extraction of brains from whole head images is included here. This software tool increases speed, can be used with an atlas or a template from within the dataset, and produces masks that need little further refinement. Conclusions: Our new automation can be applied to any MR dataset, since the starting point is a template mask generated specifically for that dataset. The method reliably and rapidly extracts brain images from whole head images, rendering them useable for subsequent analytical processing. This software tool will accelerate the exploitation of mouse models for the investigation of human brain disorders by MRI

Interpreting an apoptotic corpse as anti-inflammatory involves a chloride sensing pathway

Apoptotic cell clearance (efferocytosis) elicits an anti-inflammatory response by phagocytes, but the mechanisms that underlie this response are still being defined. Here, we uncover a chloride-sensing signalling pathway that controls both the phagocyte 'appetite' and its anti-inflammatory response. Efferocytosis transcriptionally altered the genes that encode the solute carrier (SLC) proteins SLC12A2 and SLC12A4. Interfering with SLC12A2 expression or function resulted in a significant increase in apoptotic corpse uptake per phagocyte, whereas the loss of SLC12A4 inhibited corpse uptake. In SLC12A2-deficient phagocytes, the canonical anti-inflammatory program was replaced by pro-inflammatory and oxidative-stress-associated gene programs. This 'switch' to pro-inflammatory sensing of apoptotic cells resulted from the disruption of the chloride-sensing pathway (and not due to corpse overload or poor degradation), including the chloride-sensing kinases WNK1, OSR1 and SPAK-which function upstream of SLC12A2-had a similar effect on efferocytosis. Collectively, the WNK1-OSR1-SPAK-SLC12A2/SLC12A4 chloride-sensing pathway and chloride flux in phagocytes are key modifiers of the manner in which phagocytes interpret the engulfed apoptotic corpse

Pannexin 1 channels facilitate communication between T cells to restrict the severity of airway inflammation

Allergic airway inflammation is driven by type-2 CD4(+) T cell inflammatory responses. We uncover an immunoregulatory role for the nucleotide release channel, Panx1, in T cell crosstalk during airway disease. Inverse correlations between Panx1 and asthmatics and our mouse models revealed the necessity, specificity, and sufficiency of Panx1 in T cells to restrict inflammation. Global Panx1(-/-) mice experienced exacerbated airway inflammation, and T-cell-specific deletion phenocopied Panx1(-/-) mice. A transgenic designed to re-express Panx1 in T cells reversed disease severity in global Panx1(-/-) mice. Panx1 activation occurred in pro-inflammatory T effector (Teff) and inhibitory T regulatory (Treg) cells and mediated the extracellular-nucleotide-based Treg-Teff crosstalk required for suppression of Teff cell proliferation. Mechanistic studies identified a Salt-inducible kinase-dependent phosphorylation of Panx1 serine 205 important for channel activation. A genetically targeted mouse expressing non-phosphorylatable Panx1S205A phenocopied the exacerbated inflammation in Panx1(-/-) mice. These data identify Panx1-dependent Treg:Teff cell communication in restricting airway disease

Evolution of substrate specificity in a retained enzyme driven by gene loss.

The connection between gene loss and the functional adaptation of retained proteins is still poorly understood. We apply phylogenomics and metabolic modeling to detect bacterial species that are evolving by gene loss, with the finding that Actinomycetaceae genomes from human cavities are undergoing sizable reductions, including loss of L-histidine and L-tryptophan biosynthesis. We observe that the dual-substrate phosphoribosyl isomerase A or priA gene, at which these pathways converge, appears to coevolve with the occurrence of trp and his genes. Characterization of a dozen PriA homologs shows that these enzymes adapt from bifunctionality in the largest genomes, to a monofunctional, yet not necessarily specialized, inefficient form in genomes undergoing reduction. These functional changes are accomplished via mutations, which result from relaxation of purifying selection, in residues structurally mapped after sequence and X-ray structural analyses. Our results show how gene loss can drive the evolution of substrate specificity from retained enzymes

Clinical experiences of delayed contrast enhancement with cardiac computed tomography: case series

Background: Myocardial delayed enhancement (MDE) by gadolinium-enhanced cardiac MRI is well established for myocardial scar assessment in ischemic and non-ischemic heart disease. The role of MDE by cardiac CT (CT-MDE) is not yet defined. Findings: We reviewed all clinical cases of CT-MDE at a tertiary referral center to present the cases as a case series. All clinical cardiac CT exams which utilized CT-MDE imaging between January 1, 2005 and October 1, 2010 were collected as a series and their findings were also compared with available myocardial imaging to assess for myocardial abnormalities, including echocardiography (wall motion, morphology), cardiac MRI (delayed enhancement, morphology), SPECT MPI (perfusion defects). 5,860 clinical cardiac CT exams were performed during the study period. CT-MDE was obtained in 18 patients and was reported to be present in 9 patients. The indications for CT-MDE included ischemic and non-ischemic heart diseases. In segments positive for CT-MDE, there was excellent agreement of CT with other modalities: echocardiography (n=8) demonstrated abnormal morphology and wall motion (k=1.0 and k=0.82 respectively); prior MRI (n=2) demonstrated abnormal delayed enhancement (MR-MDE) (k=1.0); SPECT MPI (n=1) demonstrated fixed perfusion defects (k=1.0). In the subset of patients without CT-MDE, no abnormal segments were identified by echocardiography (n=8), MRI (n=1) and nuclear MPI (n=0). Conclusions: CT-MDE was performed in rare clinical situations. The indications included both ischemic and non-ischemic heart disease and there was an excellent agreement between CT-MDE and abnormal myocardium by echocardiography, cardiac MRI, and nuclear MPI

Pannexin 1 drives efficient epithelial repair after tissue injury

Epithelial tissues such as lung and skin are exposed to the environment and therefore particularly vulnerable to damage during injury or infection. Rapid repair is therefore essential to restore function and organ homeostasis. Dysregulated epithelial tissue repair occurs in several human disease states, yet how individual cell types communicate and interact to coordinate tissue regeneration is incompletely understood. Here, we show that pannexin 1 (Panx1), a cell membrane channel activated by caspases in dying cells, drives efficient epithelial regeneration after tissue injury by regulating injury-induced epithelial proliferation. Lung airway epithelial injury promotes the Panx1-dependent release of factors including ATP, from dying epithelial cells, which regulates macrophage phenotype after injury. This process, in turn, induces a reparative response in tissue macrophages that includes the induction of the soluble mitogen amphiregulin, which promotes injury-induced epithelial proliferation. Analysis of regenerating lung epithelium identified Panx1-dependent induction of Nras and Bcas2, both of which positively promoted epithelial proliferation and tissue regeneration in vivo. We also established that this role of Panx1 in boosting epithelial repair after injury is conserved between mouse lung and zebrafish tailfin. These data identify a Panx1-mediated communication circuit between epithelial cells and macrophages as a key step in promoting epithelial regeneration after injury

Three red suns in the sky: A transiting, terrestrial planet in a triple M-dwarf system at 6.9 pc

We present the discovery from Transiting Exoplanet Survey Satellite (TESS) data of LTT 1445Ab. At a distance of 6.9 pc, it is the second nearest transiting exoplanet system found to date, and the closest one known for which the primary is an M dwarf. The host stellar system consists of three mid-to-late M dwarfs in a hierarchical configuration, which are blended in one TESS pixel. We use MEarth data and results from the Science Processing Operations Center data validation report to determine that the planet transits the primary star in the system. The planet has a radius of ${1.38}_{-0.12}^{+0.13}$ ${R}_{\oplus }$, an orbital period of ${5.35882}_{-0.00031}^{+0.00030}$ days, and an equilibrium temperature of ${433}_{-27}^{+28}$ K. With radial velocities from the High Accuracy Radial Velocity Planet Searcher, we place a 3σ upper mass limit of 8.4 ${M}_{\oplus }$ on the planet. LTT 1445Ab provides one of the best opportunities to date for the spectroscopic study of the atmosphere of a terrestrial world. We also present a detailed characterization of the host stellar system. We use high-resolution spectroscopy and imaging to rule out the presence of any other close stellar or brown dwarf companions. Nineteen years of photometric monitoring of A and BC indicate a moderate amount of variability, in agreement with that observed in the TESS light-curve data. We derive a preliminary astrometric orbit for the BC pair that reveals an edge-on and eccentric configuration. The presence of a transiting planet in this system hints that the entire system may be co-planar, implying that the system may have formed from the early fragmentation of an individual protostellar core.Accepted manuscrip