Efficacy of novel SPAK inhibitor ZT-1a derivatives (1c, 1d, 1g & 1h) on improving post-stroke neurological outcome and brain lesion in mice

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this recordData availability: Data will be made available on request.SPAK inhibitor ZT-1a was previously shown to be neuroprotective in murine ischemic stroke models. In this study, we further examined the efficacy of four ZT-1a derivatives (ZT-1c, -1d, -1g and -1h) on reducing stroke-induced sensorimotor function impairment and brain lesions. Vehicle control (Veh) or ZT-1 derivatives were administered via osmotic pump to adult C57BL/6J mice during 3–21 h post-stroke. Neurological behavior of these mice was assessed at days 1, 3, 5, and 7 post-stroke and MRI T2WI and DTI analysis was subsequently conducted in ex vivo brains. Veh-treated stroke mice displayed sensorimotor function deficits compared to Sham mice. In contrast, mice receiving ZT-1a derivatives displayed significantly lower neurological deficits at days 3–7 post-stroke (p -1c > -1g > -1h. The Veh-treated stroke mice displayed white matter tissue injury, reflected by reduced fractional anisotropy (FA) or axial diffusivity (AD) values in external capsule, internal capsule and hippocampus. In contrast, only ZT-1a-as well as ZT-1c-treated stroke mice exhibited significantly higher FA and AD values. These findings demonstrate that post-stroke administration of SPAK inhibitor ZT-1a and its derivatives (ZT-1c and ZT-1d) is effective in protecting gray and white matter tissues in ischemic brains, showing a potential for ischemic stroke therapy development.Veteran AffairsNational Institutes of Health (NIH)Royal Societ

In Vivo Imaging of Stepwise Vessel Occlusion in Cerebral Photothrombosis of Mice by 19F MRI

Background $^{19}$F magnetic resonance imaging (MRI) was recently introduced as a promising technique for in vivo cell tracking. In the present study we compared $^{19}$F MRI with iron-enhanced MRI in mice with photothrombosis (PT) at 7 Tesla. PT represents a model of focal cerebral ischemia exhibiting acute vessel occlusion and delayed neuroinflammation. Methods/Principal Findings Perfluorocarbons (PFC) or superparamagnetic iron oxide particles (SPIO) were injected intravenously at different time points after photothrombotic infarction. While administration of PFC directly after PT induction led to a strong $^{19}$F signal throughout the entire lesion, two hours delayed application resulted in a rim-like $^{19}$F signal at the outer edge of the lesion. These findings closely resembled the distribution of signal loss on T2-weighted MRI seen after SPIO injection reflecting intravascular accumulation of iron particles trapped in vessel thrombi as confirmed histologically. By sequential administration of two chemically shifted PFC compounds 0 and 2 hours after illumination the different spatial distribution of the $^{19}$F markers (infarct core/rim) could be visualized in the same animal. When PFC were applied at day 6 the fluorine marker was only detected after long acquisition times ex vivo. SPIO-enhanced MRI showed slight signal loss in vivo which was much more prominent ex vivo indicative for neuroinflammation at this late lesion stage. Conclusion Our study shows that vessel occlusion can be followed in vivo by $^{19}$F and SPIO-enhanced high-field MRI while in vivo imaging of neuroinflammation remains challenging. The timing of contrast agent application was the major determinant of the underlying processes depicted by both imaging techniques. Importantly, sequential application of different PFC compounds allowed depiction of ongoing vessel occlusion from the core to the margin of the ischemic lesions in a single MRI measurement

Data preparation protocol for low signal-to-noise ratio fluorine-19 MRI

Fluorine-19 MRI shows great promise for a wide range of applications including renal imaging, yet the typically low signal-to-noise ratios and sparse signal distribution necessitate a thorough data preparation.This chapter describes a general data preparation workflow for fluorine MRI experiments. The main processing steps are: (1) estimation of noise level, (2) correction of noise-induced bias and (3) background subtraction. The protocol is supplemented by an example script and toolbox available online.This chapter is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This analysis protocol chapter is complemented by two separate chapters describing the basic concept and experimental procedure

Modulation of brain cation-Cl− cotransport via the SPAK kinase inhibitor ZT-1a

This is the final version. Available on open access from Nature Research via the DOI in this recordThe SLC12A cation-Clcotransporters (CCC), including NKCC1 and the KCCs, are important determinants of brain ionic homeostasis. SPAK kinase (STK39) is the CCC master regulator, which stimulates NKCC1 ionic influx and inhibits KCC-mediated efflux via phosphorylation at conserved, shared motifs. Upregulation of SPAK-dependent CCC phosphorylation has been implicated in several neurological diseases. Using a scaffold-hybrid strategy, we develop a novel potent and selective SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide ("ZT-1a"). ZT-1a inhibits NKCC1 and stimulates KCCs by decreasing their SPAK-dependent phosphorylation. Intracerebroventricular delivery of ZT-1adecreases inflammation-induced CCC phosphorylation in the choroid plexus and reduces cerebrospinal fluid (CSF) hypersecretion in a model of post-hemorrhagic hydrocephalus. Systemically administered ZT-1a reduces ischemia-induced CCC phosphorylation, attenuates cerebral edema, protects against brain damage, and improves outcomes in a model of stroke. These results suggest ZT-1a or related compounds may be effective CCC modulators with therapeutic potential for brain disorders associated with impaired ionic homeostasis.National Key R&D ProgramNational Natural Science Foundation of ChinaFundamental Research Funds for the Central Universities of ChinaHydrocephalus FoundationSimons Foundatio

Genome Sequence of the Pea Aphid Acyrthosiphon pisum

Aphids are important agricultural pests and also biological models for studies of insect-plant interactions, symbiosis, virus vectoring, and the developmental causes of extreme phenotypic plasticity. Here we present the 464 Mb draft genome assembly of the pea aphid Acyrthosiphon pisum. This first published whole genome sequence of a basal hemimetabolous insect provides an outgroup to the multiple published genomes of holometabolous insects. Pea aphids are host-plant specialists, they can reproduce both sexually and asexually, and they have coevolved with an obligate bacterial symbiont. Here we highlight findings from whole genome analysis that may be related to these unusual biological features. These findings include discovery of extensive gene duplication in more than 2000 gene families as well as loss of evolutionarily conserved genes. Gene family expansions relative to other published genomes include genes involved in chromatin modification, miRNA synthesis, and sugar transport. Gene losses include genes central to the IMD immune pathway, selenoprotein utilization, purine salvage, and the entire urea cycle. The pea aphid genome reveals that only a limited number of genes have been acquired from bacteria; thus the reduced gene count of Buchnera does not reflect gene transfer to the host genome. The inventory of metabolic genes in the pea aphid genome suggests that there is extensive metabolite exchange between the aphid and Buchnera, including sharing of amino acid biosynthesis between the aphid and Buchnera. The pea aphid genome provides a foundation for post-genomic studies of fundamental biological questions and applied agricultural problems

Hardware considerations for preclinical magnetic resonance of the kidney

Magnetic resonance imaging (MRI) is a noninvasive imaging technology that offers unparalleled anatomical and functional detail, along with diagnostic sensitivity. MRI is suitable for longitudinal studies due to the lack of exposure to ionizing radiation. Before undertaking preclinical MRI investigations of the kidney, the appropriate MRI hardware should be carefully chosen to balance the competing demands of image quality, spatial resolution, and imaging speed, tailored to the specific scientific objectives of the investigation. Here we describe the equipment needed to perform renal MRI in rodents, with the aim to guide the appropriate hardware selection to meet the needs of renal MRI applications.This publication is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This chapter on hardware considerations for renal MRI in small animals is complemented by two separate publications describing the experimental procedure and data analysis

Functional imaging using fluorine ((19)F) MR methods: basic concepts

Kidney-associated pathologies would greatly benefit from noninvasive and robust methods that can objectively quantify changes in renal function. In the past years there has been a growing incentive to develop new applications for fluorine ((19)F) MRI in biomedical research to study functional changes during disease states. (19)F MRI represents an instrumental tool for the quantification of exogenous (19)F substances in vivo. One of the major benefits of (19)F MRI is that fluorine in its organic form is absent in eukaryotic cells. Therefore, the introduction of exogenous (19)F signals in vivo will yield background-free images, thus providing highly selective detection with absolute specificity in vivo. Here we introduce the concept of (19)F MRI, describe existing challenges, especially those pertaining to signal sensitivity, and give an overview of preclinical applications to illustrate the utility and applicability of this technique for measuring renal function in animal models. This chapter is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This introduction chapter is complemented by two separate chapters describing the experimental procedure and data analysis

Enhanced cellular uptake and long-term retention of chitosan-modified iron-oxide nanoparticles for MRI-based cell tracking

Sasha H Bakhru,1 Eda Altiok,2 Christopher Highley,1 Daniel Delubac,1 Joseph Suhan,2 T Kevin Hitchens,2 Chien Ho,2 Stefan Zappe11Department of Biomedical Engineering, 2Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USAAbstract: Tracking cells after therapeutic transplantation is imperative for evaluation of implanted cell fate and function. In this study, ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) were surface functionalized with water-soluble chitosan, a cationic polysaccharide that mediates enhanced endocytic uptake, endosomal escape into the cytosol, and subsequent long-term retention of nanoparticles. NP surface and chitosan were independently fluorescently labeled. Our NPs enable NP trafficking studies and determination of fate beyond uptake by fluorescence microscopy as well as tracking of labeled cells as localized regions of hypointensity in T2*-weighted magnetic resonance imaging (MRI) images. Adult rat neural stem cells (NSCs) were labeled with NPs, and assessment of NSC proliferation rates and differentiation potential revealed no significant differences between labeled and unlabeled NSCs. Significantly enhanced uptake of chitosan NPs in comparison to native NPs was confirmed by transmission electron microscopy, nuclear magnetic resonance (NMR) spectroscopy and in vitro cellular MRI at 11.7 Tesla. While only negligible fractions of native NPs enter cells, chitosan NPs appear within membranous vesicles within 2 hours of exposure. Additionally, chitosan-functionalized NPs escaped from membrane-bound vesicles within days, circumventing NP endo-lysosomal trafficking and exocytosis and hence enabling long-term tracking of labeled cells. Finally, our labeling strategy does not contain any NSC-specific reagents. To demonstrate general applicability across a variety of primary and immortalized cell types, embryonic mouse NSCs, mouse embryonic stem cells, HEK 293 kidney cells, and HeLa cervical cancer cells were additionally exposed to chitosan-USPIO NPs and exhibited similarly efficient loading as verified by NMR relaxometry. Our efficient and versatile labeling technology can support cell tracking with close to single cell resolution by MRI in vitro, for example, in complex tissue models not optically accessible by confocal or multi-photon fluorescence microscopy, and potentially in vivo, for example, in animal models of human disease or injury.Keywords: nanoparticle, iron oxide, chitosan, neural stem cell, cell trackin