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• W3155485089 abstract "The pathogenesis of intracerebral hemorrhage (ICH) can be divided into two distinct injury phases. Almost immediately following ICH, mechanical tissue disruption and mass effect induces primary brain injury, followed by an activated immune response to damaged brain cells and products of red blood cell lysis that drives a secondary wave of brain injury. Secondary brain injury causes increased intracranial pressure and worse pathological outcomes in both pre-clinical models and in ICH patients (Wang, 2010). Modulating the immune response represents a realistic target for therapeutics after ICH, however to date a precise understanding of the specific neuroinflammatory landscape in ICH is currently lacking. Although anti-inflammatory trials for ICH have started, these are based on observations from ischemic stroke and subarachnoid hemorrhage trials (Galea et al., 2017; BLOC-ICH, 2018; Smith et al., 2018). Using transcriptomic profiling to improve our fundamental understanding of the molecular regulation of inflammation after ICH could reveal new therapeutic strategies for patients to improve clinical outcomes after stroke.Leukocytes can contribute to both neuroprotection and neurodegeneration after ICH-induced brain injury. Following primary injury, extravasation of leukocytes into the brain parenchyma contributes to a rise in intracranial pressure and oedema. Activated innate immune cells, such as macrophages and neutrophils, are recruited from the periphery to the brain and generate free radicals that contribute to oxidative damage of neurons (Zhao et al., 2007). Conversely, activated microglia and brain macrophages, are also essential for haematoma clearance and recovery after ICH (Zhao et al., 2015). Studying the transcriptomic profile in these cell types will contribute to our understanding of how these cells are regulated following ICH and may highlight specific molecular targets to modulate the inflammatory response to benefit patients.Here, we employed a zebrafish larval model of spontaneous ICH (bubblehead; bbh) to identify a transcriptomic signature of the innate immune response in the secondary injury phase following ICH. The bbh mutant has a hypomorphic mutation in βpix, resulting in leaky neurovasculature at a critical developmental timepoint (Liu et al., 2007). Zebrafish larvae represent a useful alternative in vivo system for studying ICH pre-clinically. Unlike more commonly used rodent models, ICH in bbh mutant zebrafish larvae can occur without the need for invasive surgical procedures and thereby mimic the spontaneous nature of the human condition more closely. We have previously characterized disease outcomes in the zebrafish bbh model which recapitulates key pathological outcomes observed in mammalian models and patients (Crilly et al., 2018, 2019). The secondary injury response in zebrafish larvae is identified by an increase in macrophage recruitment and activation within the head at 24 h post injury, whilst neutrophils are the most numerous leukocyte population at this developmental stage. Using a double transgenic zebrafish reporter line crossed onto the mutant bbh background, we isolated macrophages and neutrophils from this period of secondary injury 24 h after ICH and performed transcriptomic profiling using RNA-Seq analyses (Figure 1). Data acquired in this study showed differential gene expression in leukocytes between ICH– and ICH+ groups, in addition to the differences between the two cell types (Figure 2).Open in a separate windowFigure 1Experimental workflow and quality control steps for transcriptomic analysis. (A) Diagrammatical representation of the sample acquisition and preparation. Bbh, Bubblehead mutants; mpo, myeloperoxidase; GFP, green fluorescent protein; mpeg1, macrophage expressed gene 1; mCherry, red fluorescent protein; ICH+, hemorrhaged larvae; ICH–, non-hemorrhaged sibling controls; hpf, hours post fertilization; FACS, fluorescence-activated cell sorting; RNA-Seq, RNA sequencing. (B) Representative images of ICH– and ICH+ mpo:mpeg larvae at 72 hpf. Larvae were anesthetized briefly and images were acquired using the Leica M205 FA Stereo fluorescence microscope. Larvae were confirmed to have expression of green mpo positive neutrophils and red mpeg1 positive macrophages, and separated according to presence of ICH (*). Images at higher magnification (top panels) shows an increase in mpeg1 positive cells within the brain as previously observed. (C) Sort strategy to isolate macrophages and neutrophils from ICH+ and ICH– control zebrafish larvae at 72 hpf. From a single cell suspension, neutrophils were sorted based on their expression of GFP and macrophages by their expression of mCherry. Cells expressing both (P5) were excluded from all analyses. Numbers represent frequencies of macrophages and neutrophils as a proportion of viable cells. Plots are representative from three independent replicates. (D) FastQC plots for the quality across all bases from all 12 samples. Graphical representation of the quality for each base pair position in the reads. Green region equates to very good quality calls, orange is reasonable and red poor quality. Box plots show the median value and interquartile range, whiskers include 10 and 90% points. Blue line is overall mean quality." @default.
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• W3155485089 date "2021-04-14" @default.
• W3155485089 modified "2023-09-25" @default.
• W3155485089 title "RNA-Seq Dataset From Isolated Leukocytes Following Spontaneous Intracerebral Hemorrhage in Zebrafish Larvae" @default.
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• W3155485089 doi "https://doi.org/10.3389/fncel.2021.660732" @default.
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