Animal Imaging Research Articles

Correcting for imaging gradients-related bias of T2 relaxation times at high-resolution MRI.

PurposeHigh‐resolution animal imaging is an integral part of preclinical drug development and the investigation of diseases' pathophysiology. Quantitative mapping of T2 relaxation times (qT2) is a valuable tool for both preclinical and research applications, providing high sensitivity to subtle tissue pathologies. High‐resolution T2 mapping, however, suffers from severe underestimation of T2 values due to molecular diffusion. This affects both single‐echo and multi‐echo spin echo (SSE and MESE), on top of the well‐known contamination of MESE signals by stimulated echoes, and especially on high‐field and preclinical scanners in which high imaging gradients are used in comparison to clinical scanners.MethodsDiffusion bias due to imaging gradients was analyzed by quantifying the effective b‐value for each coherence pathway in SSE and MESE protocols, and incorporating this information in a joint T2‐diffusion reconstruction algorithm. Validation was done on phantoms and in vivo mouse brain using a 9.4T and a 7T MRI scanner.ResultsUnderestimation of T2 values due to strong imaging gradients can reach up to 70%, depending on scan parameters and on the sample's diffusion coefficient. The algorithm presented here produced T2 values that agreed with reference spectroscopic measurements, were reproducible across scan settings, and reduced the average bias of T2 values from −33.5 ± 20.5% to −0.1 ± 3.6%.ConclusionsA new joint T2‐diffusion reconstruction algorithm is able to negate imaging gradient–related underestimation of T2 values, leading to reliable mapping of T2 values at high resolutions.

MIF improves immune microenvironment of Lewis lung cancer brain metastases after radiotherapy via reducing M2 macrophages.

e21026 Background: Our previous studies showed that inhibition of MIF/CD74 signaling pathway can promote the transformation of intracranial macrophages from M2 to M1 type after radiotherapy in brain metastases(BMs). It is reported that M2 macrophages are the main components of tumor immunosuppression microenvironment, and the surface marker, ARG-1, can decompose the Arginine which is essential in the growth and development of T cells. Therefore, this paper will focus on the mechanism of MIF regulating tumor infiltrating lymphocytes (TILs) by inducing macrophage phenotype transformation. Methods: MIF-Knockdown in Lewis cells were constructed by lentiviral shRNA interference technique. The expression levels of MIF in Lewis and iNOS (M1 type marker), Arg-1 (M2 type marker) in BV2 cells were detected by WB. The RNA expression levels of M1 and M2 tags were detected by qPCR. The expression of iNOS, Arg-1 and Iba-1 in lung cancer BMs model mice was detected by IF. The content of arginine in tumor tissue and clinical patient specimens were detected by Arginine kit. The proportion of TILs in vivo was measured by flow cytometry. BMs tumor growth in mice was observed by Small Animals Imaging Technology. Results: In animal experiments, WB、PCR and IF results showed that MIF knockdown (sh-MIF) combined with whole brain irradiation(IR) can raise the expression of iNOS, while the Arg-1 lessend, suggesting that downregulation of MIF can promote M1 phenotype and reduce M2 phenotype. Flow cytometry results showed that sh-MIF combined with IR increased the proportion of CD8+T/CD4+T cells . After clearance of macrophage, Flow cytometry showed no difference between the sh-MIF group and the control group (NC). Small Animals Imaging results also showed that the inhibition of tumor growth by sh-MIF was less significant than macrophages presence. It is indicated that down-regulation of MIF expression combined with irradiation promote T cell infiltration and inhibit tumor may be mediated by macrophages. Arginine kit results showed that the content of arginine increased after IR or sh-MIF, while it increased overall after macrophage clearance, but the difference between each groups was not significant, suggesting that IR or sh-MIF could increase the content of arginine by reducing M2-type macrophages. Flow cytometry and animals imaging results showed that the CD8+/CD4+TILs ratio was increased and tumor growth was inhibited after the addition of exogenous arginine. In clinical samples, the content of arginine in CSF decreased after radiotherapy, which suggests that arginine supplementation has the potential to improve the effect of radiotherapy. Conclusions: Inhibitory MIF expression can reduce the expression of Arg-1 from M2 type macrophages in BMs, thus raise the content of arginine, promote the infiltration of lymphocytes, improve the immune microenvironment, achieve radiotherapy sensitization finally.

Dual-Tuned Lattice Balun for Multi-Nuclear MRI and MRS.

Balun or trap circuits are critical components for suppressing common-mode currents flowing on the outer conductors of coaxial cables in RF coil systems for Magnetic Resonance Imaging (MRI) and Magnetic Resonance Spectroscopy (MRS). Common-mode currents affect coils' tuning and matching, induce losses, pick up extra noise from the surrounding environment, lead to undesired cross-talk, and cause safety concerns in animal and human imaging. First proposed for microwave antenna applications, the Lattice balun has been widely used in MRI coils. It has a small footprint and can be easily integrated with coil tuning/matching circuits. However, the Lattice balun is typically a single-tuned circuit and cannot be used for multi-nuclear MRI and MRS with two RF frequencies. This work describes a dual-tuned Lattice balun design that is suitable for multi-nuclear MRI/MRS. It was first analyzed theoretically to derive component values. RF circuit simulations were then performed to validate the theoretical analysis and provide guidance for practical construction. Based on the simulation results, a dual-tuned balun circuit was built for 7T 1H/23Na MRI and bench tested. The fabricated dual-tuned balun exhibits superior performance at the Larmor frequencies of both 1H and 23Na, with less than 0.15 dB insertion loss and better than 17 dB common-mode rejection ratio at both frequencies.

Imaging Capabilities of the ¹H-X-Nucleus Metamaterial-Inspired Multinuclear RF-Coil

In this paper, we present the initial experimental investigation of a two-coil receive/transmit design for small animals imaging at 7T MRI. The system uses a butterfly-type coil tuned to 300 MHz for scanning the 1H nuclei and a non-resonant loop antenna with a metamaterial-inspired resonator with the ability to tune over a wide frequency range for X-nuclei. 1H, 31P, 23Na and 13C, which are of particular interest in biomedical MRI, were selected as test nuclei in this work. Coil simulations show the two parts of the radiofrequency (RF) assembly to be decoupled and operating independently due to the orthogonality of the excited RF transverse magnetic fields. Simulations and phantom experimental imaging show sufficiently homogeneous transverse transmit RF fields and tuning capabilities for the pilot multiheteronuclear experiments.

Design and Fabrication of a High-Frequency Microconvex Array Transducer for Small Animals Imaging.

High-frequency convex array transducer, featuring both high spatial resolution and wide field of view, has been successfully developed for ophthalmic imaging. To further expand its application range to small animals' imaging, this work develops a high-frequency microconvex array transducer possessing smaller aperture size and wider scanning angle. This transducer featured 128 array elements arranged in a curvilinear 2-2 piezoelectric composite configuration, yielding a maximum view angle of 97.8°. The array was composed of two front matching layers, a nonconductive backing layer, and a customized flexible circuit that electrically connected array elements to coaxial cables. The center frequency and the -6-dB fractional bandwidth were about 18.14 MHz and 69.15%, respectively. The image of a tungsten wire phantom resulted in approximately 62.9- [Formula: see text] axial resolution and 121.3- [Formula: see text] lateral resolution. The image of the whole kidney of a rat as well as its internal arteries was acquired in vivo, demonstrating the imaging capability of the proposed high-frequency microconvex array transducers for small animals' imaging applications.

The Ever-Growing Field of High-Frequency Biomedical Ultrasound

Compared with other medical imaging counterparts, ultrasound has advantages owing to its real-time imaging capability, easy access, and cost-effectiveness. High-frequency (>20 MHz) ultrasound has been developed over the last few decades and is attractive because it provides high-resolution medical imaging and ultrasound backscattering microscopy (UBM) measurements, with applications targeting small animal imaging and some areas of human diagnosis. In recent years, driven by growing needs in intravascular ultrasound (IVUS) imaging, minimally invasive surgery, and particle manipulation, high-frequency ultrasound has continued to flourish as a niche modality and has expanded its important role in both diagnostic imaging and therapy. Concomitant needs have thus arisen in technological research and development to innovate materials, devices, algorithms, and systems for a variety of high-frequency ultrasound applications in medicine and biology.

C. elegans enteric motor neurons fire synchronized action potentials underlying the defecation motor program

C. elegans neurons were thought to be non-spiking until our recent discovery of action potentials in the sensory neuron AWA; however, the extent to which the C. elegans nervous system relies on analog or digital coding is unclear. Here we show that the enteric motor neurons AVL and DVB fire synchronous all-or-none calcium-mediated action potentials following the intestinal pacemaker during the rhythmic C. elegans defecation behavior. AVL fires unusual compound action potentials with each depolarizing calcium spike mediated by UNC-2 followed by a hyperpolarizing potassium spike mediated by a repolarization-activated potassium channel EXP-2. Simultaneous behavior tracking and imaging in free-moving animals suggest that action potentials initiated in AVL propagate along its axon to activate precisely timed DVB action potentials through the INX-1 gap junction. This work identifies a novel circuit of spiking neurons in C. elegans that uses digital coding for long-distance communication and temporal synchronization underlying reliable behavioral rhythm.

Modelling of magnetic microbubbles to evaluate contrast enhanced magnetomotive ultrasound in lymph nodes - a pre-clinical study.

Despite advances in MRI the detection and characterisation of lymph nodes in rectal cancer remains complex, especially when assessing the response to neoadjuvant treatment. An alternative approach is functional imaging, previously shown to aid characterisation of cancer tissues. We report proof of concept of the novel technique Contrast-Enhanced Magneto-Motive Ultrasound (CE-MMUS) to recover information relating to local perfusion and lymphatic drainage, and interrogate tissue mechanical properties through magnetically induced deformations. The feasibility of the proposed application was explored using a combination of experimental animal and phantom ultrasound imaging, along with finite element analysis. First, contrast-enhanced ultrasound imaging on one wild type mouse recorded lymphatic drainage of magnetic microbubbles after bolus injection. Second, tissue phantoms were imaged using MMUS to illustrate the force- and elasticity dependence of the magnetomotion. Third, the magnetomechanical interactions of a magnetic microbubble with an elastic solid were simulated using finite element software. Accumulation of magnetic microbubbles in the inguinal lymph node was verified using contrast enhanced ultrasound, with peak enhancement occurring 3.7 s post-injection. The magnetic microbubble gave rise to displacements depending on force, elasticity, and bubble radius, indicating an inverse relation between displacement and the latter two. Combining magnetic microbubbles with MMUS could harness the advantages of both techniques, to provide perfusion information, robust lymph node delineation and characterisation based on mechanical properties. (a) Lymphatic drainage of magnetic microbubbles visualised using contrast-enhanced ultrasound imaging and (b) magnetomechanical interactions between such bubbles and surrounding tissue could both contribute to (c) robust detection and characterisation of lymph nodes.

Direct bone marrow injection of human bone marrow-derived stromal cells into mouse femurs results in greater prostate cancer PC-3 cell proliferation, but not specifically proliferation within the injected femurs

BackgroundWhile prostate cancer (PCa) cells most often metastasize to bone in men, species-specific differences between human and mouse bone marrow mean that this pattern is not faithfully replicated in mice. Herein we evaluated the impact of partially humanizing mouse bone marrow with human bone marrow-derived stromal cells (BMSC, also known as "mesenchymal stem cells") on human PCa cell behaviour.MethodsBMSC are key cellular constituents of marrow. We used intrafemoral injection to transplant 5 × 105 luciferase (Luc) and green fluorescence protein (GFP) expressing human BMSC (hBMSC-Luc/GFP) into the right femur of non-obese diabetic (NOD)-severe combined immunodeficiency (scid) interleukin (IL)-2γ−/− (NSG) mice. Two weeks later, 2.5 × 106 PC-3 prostate cancer cells expressing DsRed (PC-3-DsRed) were delivered into the mice via intracardiac injection. PC-3-DsRed cells were tracked over time using an In Vivo Imaging System (IVIS) live animal imaging system, X-ray and IVIS imaging performed on harvested organs, and PC-3 cell numbers in femurs quantified using flow cytometry and histology.ResultsFlow cytometry analysis revealed greater PC-3-DsRed cell numbers within femurs of the mice that received hBMSC-Luc/GFP. However, while there were overall greater PC-3-DsRed cell numbers in these animals, there were not more PC-3-DsRed in the femurs injected with hBMSC-Luc/GFP than in contralateral femurs. A similar proportion of mice in with or without hBMSC-Luc/GFP had bone lessions, but the absolute number of bone lesions was greater in mice that had received hBMSC-Luc/GFP.ConclusionPC-3-DsRed cells preferentially populated bones in mice that had received hBMSC-Luc/GFP, although PC-3-DsRed cells not specifically localize in the bone marrow cavity where hBMSC-Luc/GFP had been transplanted. hBMSC-Luc/GFP appear to modify mouse biology in a manner that supports PC-3-DsRed tumor development, rather than specifically influencing PC-3-DsRed cell homing. This study provides useful insights into the role of humanizing murine bone marrow with hBMSC to study human PCa cell biology.

Growth factor dependency in mammary organoids regulates ductal morphogenesis during organ regeneration

Signaling pathways play an important role in cell fate determination in stem cells and regulate a plethora of developmental programs, the dysregulation of which can lead to human diseases. Growth factors (GFs) regulating these signaling pathways therefore play a major role in the plasticity of adult stem cells and modulate cellular differentiation and tissue repair outcomes. We consider murine mammary organoid generation from self-organizing adult stem cells as a tool to understand the role of GFs in organ development and tissue regeneration. The astounding capacity of mammary organoids to regenerate a gland in vivo after transplantation makes it a convenient model to study organ regeneration. We show organoids grown in suspension with minimal concentration of Matrigel and in the presence of a cocktail of GFs regulating EGF and FGF signaling can recapitulate key epithelial layers of adult mammary gland. We establish a toolkit utilizing in vivo whole animal imaging and ultrasound imaging combined with ex vivo approaches including tissue clearing and confocal imaging to study organ regeneration and ductal morphogenesis. Although the organoid structures were severely impaired in vitro when cultured in the presence of individual GFs, ex vivo imaging revealed ductal branching after transplantation albeit with significantly reduced number of terminal end buds. We anticipate these imaging modalities will open novel avenues to study mammary gland morphogenesis in vivo and can be beneficial for monitoring mammary tumor progression in pre-clinical and clinical settings.

Prevention of Type 1 Diabetes via a Lipid Nanoparticle-Based Oral Immunotherapy that Targets Selective Lymphoid Tissues

Abstract Type 1 diabetes (T1D) is an untreatable autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. Antigen presenting cells (APCs) malfunctions and aberrant accumulation of type 1 interferons contribute to T1D, and the inhibition of inflammatory cytokines signaling via JAK inhibitors like Tofacitinib (Tofa) has shown promise in the prevention of the disease. However, maintenance of a therapeutic concentration of these drugs is challenging and their prolonged use leads to significant side effects. Our objective was to design a controlled and localized delivery of Tofa via lipid nanoparticles (LNp) and assess the impact of an oral short-term administration strategy on T1D development. We identified a LNp formulation with negligible toxicity, readily taken up by multiple immune cells, and with favorable Tofa encapsulation efficiency. Live animal imaging confirmed these particles have the unique property of accumulating in lymphoid tissues, particularly in pancreatic and mesenteric lymph nodes when administrated via oral gavage. Importantly, short-term administration (5 gavages once every other day) of Tofa-LNp early (3-week-old) or late (10-week-old) in disease prone NOD mice promoted a significant delay of T1D onset. Cell profiling and in vivo challenges indicated that this therapeutic effect is likely due to localized inhibition of APCs maturation and, surprisingly, promotion of anergy in diabetogenic T cells. Ex-vivo stimulation confirmed the hyporeactivity of these cells and adoptive transfer experiments confirmed their ability to act as reservoir of induced regulatory T cells. These results highlight the versatility of this novel LNp-based drug delivery strategy. This work was supported by grant 2-SRA-2016-310-S-B from the Juvenile Diabetes Research Foundation.

VEGFR2 signals with VE‐cadherin and PECAM to facilitate leukocyte transendothelial migration

The migration of leukocytes out of the blood and into tissue is a critical component of the inflammatory process. To gain access to the target tissue and exert their beneficial or deleterious effects, leukocytes undergo a complex series of adhesive interactions with endothelial cells, which line the vessel wall. In the putative committed step in this process, the leukocyte must find the junction between adjacent endothelial cells and crawl through. This process, commonly referred to as transendothelial migration (TEM), has long been known to require homophilic interaction between PECAM on leukocyte and the endothelial cells. Recently our group showed that the process also involves calcium signaling originating through the cation channel TRPC6.We hypothesized that PECAM may be functioning in TEM using some of the interactions recently shown to be involved in its role in the mechanotransduction of fluid sheer stress. Perhaps engagement of leukocyte PECAM with endothelial cell PECAM at the cell border provides the sheer stress in this case. Here we report the unexpected role of a macromolecular PECAM complex that facilitates signaling to TRPC6 in TEM. Specifically, we show that endothelial cell PECAM binds to VEGFR2 and VE‐cadherin via the transmembrane domain of VE‐cadherin. Swapping the VE‐cadherin transmembrane domain with that of N‐cadherin abrogated TEM. Likewise, deletion of the cytoplasmic c‐terminal tail of PECAM also abolished this signaling cascade. Stimulation of this complex with antibodies against PECAM induces the phosphorylation of VEGFR2 on tyrosine Y1175. Mutation of that critical residue to phenylalanine (Y1175F) prevented VEGFR2 phosphorylation and blocked TEM. Furthermore we show that this complex signals specifically to PLC1γ (and not PLCβ3) leading to its phosphorylation. Blocking its function with the inhibitor U73122 also blocked TEM. Diacylglycerol liberated by PLCγ1 activates TRPC6.We also confirmed the role of VEGFR2 in vivo using to separate models of inflammation. First, we examined the recruitment of neutrophils to inflamed skin using the croton oil dermatitis model. In control animals, neutrophils are robustly recruited to and migrate across post‐capillary venules. In VEGFR2 knockout (KO) mice, most neutrophils are found inside the vessel with a corresponding decrease in those found out in tissue. Similarly, live animal imaging (intravital microscopy) of the inflamed cremaster muscle showed no defect in neutrophil rolling or adhesion in the VEGFR2 KO animals, but a profound disruption in their ability to undergo TEM compared to control wild type mice.Taken together, these data confirm the role of a VEGFR2/PECAM/VE‐cadherin mechanotransduction complex that signals through PLC1γ to facilitate leukocyte TEM upstream of the action of TRPC6. Uncovering this signaling pathway opens up several new targets for potential therapeutic intervention.

Application of Advanced Imaging Modalities in Veterinary Medicine: A Review.

Veterinary anatomy has traditionally relied on detailed dissections to produce anatomical illustrations, but modern imaging modalities, now represent an enormous resource that allows for fast non-invasive visualizations in living animals for clinical and research purposes. In this review, advanced anatomical imaging modalities and their applications, safety issues, challenges, and future prospects of the techniques commonly employed for animal imaging would be highlighted. The quality of diagnostic imaging equipment in veterinary practice has greatly improved. Recent advances made in veterinary advanced imaging specifically about cross-sectional modalities (CT and MRI), nuclear medicine (PET, SPECT), and dual imaging modalities (PET/CT, PET/MR, and SPECT/CT) have become widely available, leading to greater demands and expectations from veterinary clients. These modalities allow for the creation of three-dimensional representations that can be of considerable value in the dissemination of clinical diagnosis and anatomical studies. Despite, the modern imaging modalities well established in developed countries across the globe, it is yet to remain in its infancy stage in veterinary practice in developing countries due to heavy initial investment and maintenance costs, lack of expert interpretation, a requirement of specialized technical staff and need of adjustable machines to accommodate the different range of animal sizes. Therefore, veterinarians should take advantage of these imaging techniques in designing future experiments by considering the availability of these varied imaging modalities and the creation of three-dimensional graphical representations of internal structures.

Tomographic Imaging by a Si/CdTe Compton Camera for ¹¹¹In and ¹³¹I Radionuclides

Tomographic imaging with radionuclides commonly used in nuclear medicine, such as $^{111}$In (171 and 245 keV) and $^{131}$I (364 keV), is in high demand for medical applications and small animal imaging. The Si/CdTe Compton camera with its high angular and high energy resolutions is an especially promising detector to extend the energy coverage for imaging to the range that covers gamma-ray emitted from these radionuclides. Here, we take the first steps towards short-distance imaging by conducting experiments using three-dimensional phantoms composed of multiple sphere-like solutions of $^{111}$In and $^{131}$I with a diameter of 2.7 mm, placed at a distance of 41 mm. Using simple back-projection methods, the positions of the sources are reproduced with a spatial resolution of 11.5 mm and 9.0 mm (FWHM) for $^{111}$In and $^{131}$I, respectively. We found that a LM-MLEM method gives a better resolution of 4.0 mm and 2.7 mm (FWHM). We resolve source positions of a tetrahedron structure with a source-to-source separation of 28 mm. These findings demonstrate that Compton Cameras have the potential of close-distance imaging of radioisotopes distributions in the energy range below 400 keV.

A general technique for the estimation of farm animal body part weights from CT scans and its applications in a rabbit breeding program

Various applications of farm animal imaging are based on estimating the weight of specific body parts and cuts from CT images of animals. In many cases, the complexity of the problem is increased by the enormous variability in body postures in the CT images due to scanning unsedated live animals. In this work, we propose a general and robust approach to estimate the weights of sections and body parts from CT images of (possibly) live animals. We adapt the segmentation by elastic registration and joint feature and model selection for the regression component to cope with the large number of features and small number of samples. The proposed technique is evaluated and illustrated by real applications in rabbit breeding programs. The r2 values are 12% higher than those of previous techniques and methods used in the breeding selection programs. The proposed technique is easily transferable to similar problems and is therefore made available in an open source software package for the benefit of the community.

Scintillation event localization in hemi-ellipsoid detector for SPECT, a simulation study using Geant4 Monte-Carlo

Purpose: a high sensitivity Cardiac SPECT system using curved crystals with pinhole collimation was proposed previously (Dey, IEEE Trans. Nucl. Sci. 59 (2012) 334). Using hemi-ellipsoid CsI crystals, in simulations, the system improved sensitivity by a factor of 3 over newer generation SPECT systems (DSPECT or GE Discovery) while keeping the resolution comparable (Bhusal et al., Med. Phys. 46 (2018) 116). In this work, we hypothesize that the high curvature detector results in measurable differences in light distribution from events at different depths in the crystal. We rigorously test this by analyzing the scintillation light using Monte-Carlo (Geant4) and propose a statistical event localization method in a hemi-ellipsoid detector. We evaluate the localization error of the algorithm and the back-projected errors in object space. Methods: to develop this localization capability for the proposed design, we used Geant4 to simulate the propagation of scintillation light in a monolithic hemi-ellipsoidal CsI crystal. A look-up table (LUT) was created to map the points inside the crystal to the expected light pattern on the crystal surface using the Geant4 simulation data. Thirteen zones were considered across the crystal. In each zone, gamma-rays were simulated and the resulting photon intensity on the surface was captured, serving as our experimental interactions. An algorithm based on Poisson statistics was developed to limit the search of the experimental gamma-ray event locations into smaller regions of the LUT. The localization was fine-tuned by comparing the light distribution of the gamma interactions in selected patterns from the LUT points and then recorded. The algorithm-localized gamma-ray events were also individually back-projected to the object mid-plane, (expected mid-plane of the heart), and the error at the plane was recorded as well. Results: the light patterns of adjacent LUT points showed visually discernible differences. Excluding some outliers (up to 2%), the localized errors averaged over all the zones was 0.71 (±0.44) mm with a worse case of 1.36 (± 0.67) mm at the apex. Moreover, when back-projected to the midplane of the region of interest for Cardiac SPECT, the errors were <1 mm due to the high system magnification afforded by the apex and other zones. The average back-projection error at the mid-plane of the object was 0.4 mm±0.22 mm. Conclusion: we modeled gamma-event interactions and scintillation light spread in CsI hemi-ellipsoid detector and developed a robust statistical algorithm that localized scintillation events to within 0.71 (±0.44) mm on the average within a hemi-ellipsoid CsI detector. Moreover, due to the high system magnification afforded by the crystal, the hemi-ellipsoid unit was capable of achieving <1 mm average localization in the object space, assuming perfect pinhole collimator resolution recovery. Thus, we show that this high sensitivity system will be able to deliver images with high resolution for Cardiac SPECT. In the future, the application of this may be extended to Brain SPECT and small animal imaging.

Delivery of TAPI‐1 via Neutrophil Membrane Fusogenic Liposomal Nanoparticles Can Effectively Ameliorate Cardiac Dysfunction in Heart Failure

Nanomedicine has been widely employed as a site‐specific and target‐oriented drug delivery tactic in treating heart failure (HF). Ischemia‐induced inflammation plays a robust role in the development of HF and neutrophil is one of the main immune cell types that rapidly infiltrate into the inflamed tissues after myocardial infarction (MI). In the present study, we examined the efficacy of a formation of biomimetic nanoparticles that were prepared by fabricated TAPI‐1 loaded liposomes coated with neutrophil membranes in the treatment of HF. Male SD rats underwent coronary artery ligation (CL) to induce HF. These HF rats were treated with intravenous injection of TAPI‐1‐loaded biomimetic nanoparticles (n=6) or TAPI‐1 alone (n=6) for 4 weeks. The left ventricular (LV) function was assessed by echocardiography within 24 hours and 4 weeks after CL. The nanoparticles were evaluated prior to the treatments. Compared with non‐coated formation, neutrophil membrane‐coated nanoparticles have high uptake into LPS‐stimulated cardiac cells or endothelial cells. Moreover, the coated nanoparticles exhibited higher cell‐specific targeting potential and internalization capacity than non‐coated nanoparticles in LPS‐stimulated endothelial cells. Small animal imaging showed that DiR‐labeled nanoparticles were mainly accumulated in the injured heart after MI. Compared with HF rats treated with TAPI‐1 alone, the HF rats treated with TAPI‐1‐loaded nanoparticles had significantly (* p&lt;0.05) improved anatomic indicators with reduced ratio (%) of heart/body weight (0.34 ± 0.042* vs. 0.48± 0.11) and lung/body weight (0.84 ± 0.19* vs. 1.14 ± 0.15). Additionally, one 4‐week treatment with TAPI‐1‐loaded biomimetic nanoparticles reduced LV end systolic volume (0.67±0.032* vs. 0.83±0.036, mL) when compared with the TAPI‐1 alone. While LV stroke volume (LVSV) did not change significantly in IV TAPI‐1‐treated HF animals, LVSV (0.47±0.043* vs. 0.27±0.029, mL) was significantly increased in HF rats treated with TAPI‐1‐loaded nanoparticles at 4 weeks vs. 24 hours after CL. LV end diastolic volume, LV ejection fraction, and LV infarction zone didn’t exhibit significant differences between two treatment groups. Cardiac hemodynamic measurements revealed that the HF rats treated with TAPI‐1‐loaded nanoparticles had decreased LV end diastolic pressure (10.53±1.67* vs. 19.01±3.32, mmHg) and elevated dP/dtmax(8202.65±658.09* vs. 5994.47±630.81) compared with the HF rats‐treated with TAPI‐1 alone. Histological evaluation indicated that the fibrosis areas surrounding the infarction scar were significant reduced (15.97±1.84* vs. 22.86±2.27, %) in HF rats treated with TAPI‐1‐loaded nanoparticles vs. TAPI alone. Flow cytometric analysis also demonstrated that treatment with TAPI‐1‐loaded nanoparticles improved inflammatory conditions indicated by increased ratio of CD4+/CD8+ T Cells (2.40±0.67* vs. 1.56±0.21) in the blood and reduced percentage of leukocytes in the heart of HF. These data indicate that neutrophil membrane fusogenic liposomal nanoparticle is an effective drug delivery approach in treating this devastating disease. (Supported by NIH grants R01 HL‐139521 &amp; HL‐155091 to SGW, S10 OD019941 to RW)

Conducted Capillary Signaling Enables Oxygen Responses in Skeletal Muscle Independent of Metabolite Production

Red blood cell (RBC) flow through the microvasculature is closely matched to tissue O2 requirements. At a fundamental level, O2 demand‐supply coupling entails the sensing of PO2, and the generation of a stimulus that alters upstream arteriolar tone. However, where and how O2 needs are sensed in the microcirculation is presently contested. One idea centers on hypoxia triggering the release of K+, activating endothelial KIR2.1 channels and initiating a hyperpolarization that conducts upstream via gap junctions, comprised of connexins (Cx). We tested this idea in skeletal muscle where we controlled the local tissue O2 environment using live animal imaging in Cx40‐/‐and endothelial KIR2.1‐/‐ mice. The extensor digitorum longus muscle positioned overtop a gas control chamber allowed second‐by‐second monitoring of capillary RBC flow responses as O2 was altered around its physiological set point of 53 mmHg. A stepwise drop in PO2 at the muscle surface (53 to 15 or 0 mmHg) increased RBC supply rate in control capillaries while elevated chamber O2 elicited the opposite response; these capillaries robustly expressed Cx40. The RBC flow responses were rapid and tightly coupled to O2 levels as readily observed when chamber O2 was oscillated in a sinusoidal manner. In contrast, this blood flow response was significantly diminished in Cx40‐/‐ mice and translated into lower capillary RBC O2 saturation. KIR2.1‐/‐mice, on the other hand, had normal resting RBC O2 saturation and reacted normally to O2 changes, albeit an oscillation or a sustained stepwise decrease. Furthermore, we confirmed that RBC flow responses are conserved in endothelial KIR2.1‐/‐mice even when the low O2 challenge is applied to a restricted number of surface capillaries in the muscle; interestingly, these responses were dominated by capillary hematocrit changes in both control and endothelial KIR2.1‐/‐mice. Modelling this phenomenon supported the idea that the number of capillaries stimulated is indeed tied to conduction distance and tissue RBC distribution. In conclusion, we demonstrate that microvascular O2 responses depend on coordinated electrical signaling via gap junctions comprised of Cx40 and that endothelial KIR2.1channels do not drive the initiating electrical event. These findings reconceptualize our understanding of blood flow regulation and how O2 initiates this process at the capillary level independent of metabolite production.

Study on molecular mechanism of benzo (ɑ) pyrene on CMA by HSP90ɑ and HIF-1ɑ

ObjectiveThe effects of benzo (α) pyrene (BaP) on chaperone mediated autophagy (CMA) through heat shock protein 90 (HSP90) and hypoxia- inducible factor-1 (HIF-1) are studied by RNA interference and subcutaneous tumor formation technique in nude mice. Methods40 nude mice that were inoculated with the silenced HSP90ɑ A549 cells line under the armpits of the forelimbs were divided into 4 groups, and were intragastrically administered with 1.80 mg/kg/d BaP-corn oil solutionfor for 60d (except the Control group), and the growth curves of nude mice and transplanted tumors were recorded. The size and morphological changes of tumors were observed by small animal imaging technique. qPCR, Western blot and Immunohistochemistry were used to detect the expression of HSP90ɑ, HSC70 and Lamp-2A. A549 cells were treated with 0.1 μmol/L, 1 μmol/L and 10 μmol/L BaP for 24 h, EPO and HIF-1ɑ concentration and HIF-1ɑ protein expression were detected by Elisa and Western blot; A549 cells were treated with 10 μmol/L BaP and HIF-1ɑ inhibitor for 24 h, qPCR, Western blot and Immunofluorescence methods were used to detect the expression of HSP90ɑ, HSC70 and Lamp-2A. ResultsThe weight of nude mice and transplanted tumors silenced HSP90ɑ was reduced by BaP; the expression of HSP90ɑ, HSC70, Lamp-2A mRNA and proteins in transplanted tumor tissues silenced HSP90ɑ were reduced by BaP; the total number of bioluminescence photons of transplanted tumors silenced HSP90ɑ were reduced by BaP. The concentration of EPO and HIF-1ɑ and the expression of HIF-1ɑ protein in A549 cells was increased by 10 μmol/L BaP; with HIF-1ɑ inhibitors treated, HSP90ɑ, HSC70, Lamp-2A mRNA and proteins expression and the fluorescence intensity of HSP90ɑ were decreased of A549 cells. ConclusionsThe growth of transplanted tumor in nude mice is promoted by BaP, and is inhibited when HSP90ɑ was silenced. BaP promotes the occurrence of CMA by promoting the expression of HSP90ɑ and HIF-1ɑ, which are vital regulatory genes of BaP activation of CMA.

In-ovo imaging using ostrich eggs: Biomagnetism for detection of cardiac signals and embryonal motion.

In-ovo imaging using ostrich eggs has been described as a potential alternative to common animal testing. The main advantage is its independence from small animal imaging devices as ostrich eggs provide good image quality on regular CT, MRI, or PET used in examinations of humans. However, embryonal motion during dynamic imaging studies produce artifacts. The aims of this study were (1) to explore the feasibility of biomagnetism to detect cardiac signals and embryonal motion and to use these findings (2) to investigate the effect of isoflurane anesthesia on ostrich embryos. A standard magnetoencephalography developed for brain studies was used to detect embryonal signals of ostrich eggs on developmental day 34. Signals were instantly shown on a screen and data were also postprocessed. For assessing the effects of anesthesia, nine ostrich eggs were investigated using isoflurane 6% for 90 min. Biomagnetic signals were recorded simultaneously. A control group consisting of eight different ostrich eggs was also investigated. Cardiac signals similar to electrocardiography were observed in all eggs. Postprocessing revealed frequent motion of embryos without anesthesia. The exposure to isoflurane led to a significant decrease in motion signals in 9/9 ostrich embryos after 8 min. Motion was significantly reduced in the isoflurane group versus control group. There were no isoflurane-related deaths. This study shows that biomagnetism is feasible to detect cardiac signals and motion of ostrich embryos in-ovo. Application of isoflurane is safe and leads to a rapid decrease in embryonal motion, which is an important prerequisite for the implementation of in-ovo imaging using ostrich eggs.