Whole-mount Immunofluorescence Staining Research Articles

Baicalin administration could rescue high glucose-induced craniofacial skeleton malformation by regulating neural crest development.

Hyperglycemia in pregnancy can increase the risk of congenital disorders, but little is known about craniofacial skeleton malformation and its corresponding medication. Our study first used meta-analysis to review the previous findings. Second, baicalin, an antioxidant, was chosen to counteract high glucose-induced craniofacial skeleton malformation. Its effectiveness was then tested by exposing chicken embryos to a combination of high glucose (HG, 50mM) and 6μM baicalin. Third, whole-mount immunofluorescence staining and in situ hybridization revealed that baicalin administration could reverse HG-inhibited neural crest cells (NCC) delamination and migration through upregulating the expression of Pax7 and Foxd3, and mitigate the disordered epithelial-mesenchymal transition (EMT) process by regulating corresponding adhesion molecules and transcription factors (i.e., E-cadherin, N-cadherin, Cadherin 6B, Slug and Msx1). Finally, through bioinformatic analysis and cellular thermal shift assay, we identified the AKR1B1 gene as a potential target. In summary, these findings suggest that baicalin could be used as a therapeutic agent for high glucose-induced craniofacial skeleton malformation.

Regulation of YAP Promotor Accessibility in Endothelial Mechanotransduction.

Endothelial cells are constantly exposed to mechanical forces in the form of fluid shear stress, extracellular stiffness, and cyclic strain. The mechanoresponsive activity of YAP (Yes-associated protein) and its role in vascular development are well described; however, whether changes to transcription or epigenetic regulation of YAP are involved in these processes remains unanswered. Furthermore, how mechanical forces are transduced to the nucleus to drive transcriptional reprogramming in endothelial cells is poorly understood. The YAP target gene, AmotL2 (angiomotin-like 2), is a junctional mechanotransducer that connects cell-cell junctions to the nuclear membrane via the actin cytoskeleton. We applied mechanical manipulations including shear flow, stretching, and substrate stiffness to endothelial cells to investigate the role of mechanical forces in modulating YAP transcription. Using in vitro and in vivo endothelial depletion of AmotL2, we assess nuclear morphology, chromatin organization (using transposase-accessible chromatin sequencing), and whole-mount immunofluorescent staining of the aorta to determine the regulation and functionality of YAP. Finally, we use genetic and chemical inhibition to uncouple the nuclear-cytoskeletal connection to investigate the role of this pathway on YAP transcription. Our results reveal that mechanical forces sensed at cell-cell junctions by the YAP target gene AmotL2 are directly involved in changes in global chromatin accessibility and activity of the histone methyltransferase EZH2, leading to modulation of YAP promotor activity. Functionally, shear stress-induced proliferation of endothelial cells in vivo was reliant on AmotL2 and YAP/TAZ (transcriptional coactivator with PDZ-binding motif) expression. Mechanistically, uncoupling of the nuclear-cytoskeletal connection from junctions and focal adhesions led to altered nuclear morphology, chromatin accessibility, and YAP promotor activity. Our findings reveal a role for AmotL2 and nuclear-cytoskeletal force transmission in modulating the epigenetic and transcriptional regulation of YAP to maintain a mechano-enforced positive feedback loop of vascular homeostasis. These findings may offer an explanation as to the proinflammatory phenotype that leads to aneurysm formation observed in AmotL2 endothelial deletion models.

The Secreted Ly6/uPAR-Related Protein 1 (Slurp1) Modulates Corneal Angiogenic Inflammation Via NF-κB Signaling.

Previously we demonstrated that the secreted Ly-6/uPAR related protein 1 (SLURP1), abundantly expressed in the corneal epithelium (CE) and secreted into the tear fluid, serves as an antiangiogenic molecule. Here we describe the Slurp1-null (Slurp1X-/-) mouse corneal response to silver nitrate (AgNO3) cautery. Five days after AgNO3 cautery, we compared the wild-type (WT) and Slurp1X-/- mouse (1) corneal neovascularization (CNV) and immune cell influx by whole-mount immunofluorescent staining for CD31 and CD45, (2) macrophage and neutrophil infiltration by flow cytometry, and (3) gene expression by quantitative RT-PCR. Quantitative RT-PCR, immunofluorescent staining, and immunoblots were employed to evaluate the expression, phosphorylation status, and subcellular localization of NF-κB pathway components. Unlike the WT, the Slurp1X-/- corneas displayed denser CNV in response to AgNO3 cautery, with more infiltrating macrophages and neutrophils and greater upregulation of the transcripts encoding VEGFA, MMP2, IL-1b, and vimentin. At 2, 7, and 10days after AgNO3 cautery, Slurp1 expression was significantly downregulated in the WT corneas. Compared with the WT, naive Slurp1X-/- CE displayed increased phosphorylation of IKK(a/b), elevated phosphorylation of IκB with decreased amounts of total IκB, and higher phosphorylation of NF-κB, suggesting that NF-κB signaling is constitutively active in naive Slurp1X-/- corneas. Enhanced angiogenic inflammation in AgNO3 cauterized Slurp1X-/- corneas and constitutively active status of NF-κB signaling in the absence of Slurp1 suggest that Slurp1 modulates corneal angiogenic inflammation via NF-κB signaling.

Characterization of the Newborn Epidermis and Adult Hair Follicles Using Whole-Mount Immunofluorescent Staining of Mouse Dorsal Skin.

The mammalian integumentary system, including skin and its appendages, serves as a protective barrier for the body. During development, skin epidermis undergoes rapid cell division and differentiation to form multiple stratified layers of keratinocytes. Concurrently the epidermis also gives rise to hair follicles that invaginate into the dermis. In adult skin, the hair follicle undergoes cyclic regeneration fueled by hair follicle stem cells located in the bulge. Three-dimensional and high-resolution imaging of these structures using whole-mount immunofluorescent staining allows to better characterize epidermal progenitors and stem cells.

Whole-Mount Immunofluorescence Staining to Visualize Cell Cycle Progression in Mouse Oocyte Meiosis.

Whole-mount immunofluorescence allows direct visualization of the cellular architecture within cells. Here, we apply this technique to mouse oocytes to visualize spindle morphology and microtubule attachments to kinetochores, using a technique we call "cold treatment," at various phases of the meiotic cell cycle. This method allows the analysis of spindle structures at different meiosis I stages and at metaphase II. An adaptation of the protocol to the cell cycle stage of interest is described.

Single-Cell Transcriptomics Reveals Cellular Heterogeneity and Complex Cell-Cell Communication Networks in the Mouse Cornea.

To generate a single-cell RNA-sequencing (scRNA-seq) map and construct cell-cell communication networks of mouse corneas. C57BL/6 mouse corneas were dissociated to single cells and subjected to scRNA-seq. Cell populations were clustered and annotated for bioinformatic analysis using the R package "Seurat." Differential expression patterns were validated and spatially mapped with whole-mount immunofluorescence staining. Global intercellular signaling networks were constructed using CellChat. Unbiased clustering of scRNA-seq transcriptomes of 14,732 cells from 40 corneas revealed 17 cell clusters of six major cell types: nine epithelial cell, three keratocyte, two corneal endothelial cell, and one each of immune cell, vascular endothelial cell, and fibroblast clusters. The nine epithelial cell subtypes included quiescent limbal stem cells, transit-amplifying cells, and differentiated cells from corneas and two minor conjunctival epithelial clusters. CellChat analysis provided an atlas of the complex intercellular signaling communications among all cell types. We constructed a complete single-cell transcriptomic map and the complex signaling cross-talk among all cell types of the cornea, which can be used as a foundation atlas for further research on the cornea. This study also deepens the understanding of the cellular heterogeneity and heterotypic cell-cell interaction within corneas.

Innervation of the developing kidney in vivo and in vitro.

Within the adult kidney, renal neurites can be observed alongside the arteries where they play a role in regulating blood flow. However, their role and localization during development has so far not been described in detail. In other tissues, such as the skin of developing limb buds, neurons play an important role during arterial differentiation. Here, we aim to investigate whether renal nerves could potentially carry out a similar role during arterial development in the mouse kidney. In order to do so, we used whole-mount immunofluorescence staining to identify whether the timing of neuronal innervation correlates with the recruitment of arterial smooth muscle cells. Our results show that neurites innervate the kidney between day 13.5 and 14.5 of development, arriving after the recruitment of smooth muscle actin-positive cells to the renal arteries.

Skin Whole-Mount Immunofluorescent Staining Protocol, 3D Visualization, and Spatial Image Analysis.

The use of polychromatic immunofluorescent staining on whole-mount skin enables cell type characterization and aids in the delineation of the physiological and immunological strategies used by the skin to combat pathogens. Using whole-mount skin for polychromatic immunofluorescent staining removes the need for histological sectioning and enables the visualization of anatomical structures and immune cell types in three dimensions. Here we present a detailed protocol for immunostaining with fluorescence-conjugated primary antibodies in whole-mount skin to reveal structural landmarks and specific immune cell types using confocal laser scanning microscopy (CLSM) (Basic Protocol 1). The optimized staining panel reveals structural features such as blood vessels (CD31 antibody) and the lymphatic network (LYVE-1 antibody), in combination with MHCII antibodies for antigen-presenting cells (APCs), CD64 for macrophages and monocytes, CD103 for dendritic epidermal T cells (DETC), and CD326 for Langerhans cells (LC). Basic Protocol 2 describes image visualization pipelines using open-source software (ImageJ/FIJI), enabling four visualization options (z-projections, orthogonal views, 3D visualization, and animation). Basic Protocol 3 describes a quantitative analysis pipeline using CellProfiler to characterize the spatial relationship between cell types using mathematical indices such as Spatial Distribution Index (SDI), Neighborhood Frequency (NF), and Normalized Median Evenness (NME). These protocols will enable researchers to stain, record, analyze, and interpret data from whole-mount skin using commercially available reagents in a CLSM-equipped laboratory and freely available analysis software. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescent staining and imaging for whole-mount mouse skin Basic Protocol 2: File rendering and visualization using FIJI Basic Protocol 3: Spatial image analysis using CellProfiler.

Topical Decorin Reduces Corneal Inflammation and Imparts Neuroprotection in a Mouse Model of Benzalkonium Chloride-induced Corneal Neuropathy.

We evaluated the neuroprotective and immunomodulatory effects of topical decorin in a murine model of benzalkonium chloride (BAK)-induced corneal neuropathy. Topical BAK (0.1%) was administered daily to both eyes of female C57BL/6J mice (n = 14) for 7 days. One group of mice received topical decorin (1.07 mg/mL) eye drops to one eye and saline (0.9%) to the contralateral eye; the other group received saline eye drops to both eyes. All eye drops were given three times daily over the experimental period. A control group (n = 8) received daily topical saline only, instead of BAK. Optical coherence tomography imaging was performed before (at day 0) and after (day 7) treatment to evaluate the central corneal thickness. Whole-mount immunofluorescence staining was performed to evaluate the density of corneal intraepithelial nerves and immune cells. BAK-exposed eyes showed corneal epithelial thinning, infiltration of inflammatory macrophages and neutrophils, and a lower density of intraepithelial nerves. No change to the corneal stromal thickness or dendritic cell density was observed. After BAK exposure, decorin-treated eyes had a lower density of macrophages and less neutrophil infiltration and a higher nerve density than the saline-treated group. Contralateral eyes from the decorin-treated animals showed fewer macrophages and neutrophils relative to saline-treated animals. A negative correlation was found between corneal nerve density and macrophage or neutrophil density. Topical decorin provides neuroprotective and anti-inflammatory effects in a chemical model of BAK-induced corneal neuropathy. The attenuation of corneal inflammation by decorin may contribute to decreasing corneal nerve degeneration induced by BAK.

Erratum: Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse.

An erratum was issued for:Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse. The Authors section was updated from: Ruibing Xia1,2,3 Julia Vlcek1,2 Julia Bauer1,2,3 Stefan Kääb1,3 Hellen Ishikawa-Ankerhold1,2 Dominic Adam van den Heuvel1,2 Christian Schulz1,2,3 Steffen Massberg1,2,3 Sebastian Clauss1,2,3 1University Hospital Munich, Department of Medicine I, Ludwig Maximilian University Munich 2Walter Brendel Center of Experimental Medicine, Ludwig Maximilian University Munich 3German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance to: Ruibing Xia1,2,3 Julia Vlcek1,2 Julia Bauer1,2,3 Stefan Kääb1,3 Hellen Ishikawa-Ankerhold1,2 Dominic Adam van den Heuvel1,2 Christian Schulz1,2,3 Steffen Massberg1,2,3 Sebastian Clauss1,2,3 1University Hospital Munich, Department of Medicine I, Ludwig Maximilians University (LMU) Munich 2Institute of Surgical Research at the Walter Brendel Center of Experimental Medicine, University Hospital Munich, Ludwig Maximilians University (LMU) Munich 3German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance.

An Innovative Toolkit to Investigate the Complex Mechanisms of Cardiac Arrhythmias

ARTICLES DISCUSSED: Tomsits, P., Schuttler, D., Kaab, S., Clauss, S., Voigt, N. Isolation of high quality murine atrial and ventricular myocytes for simultaneous measurements of Ca2+ transients and L-Type calcium current. Journal of Visualized Experiments. (165), 10.3791/61964 (2020). Seibertz, F., Reynolds, M., Voigt, N. Single-Cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes. Journal of Visualized Experiments. (166), 10.3791/61890 (2020). Xia, R. et al. Isolation and culture of resident cardiac macrophages from the murine sinoatrial and atrioventricular node. Journal of Visualized Experiments. (171), 10.3791/62236 (2021). Xia, R. et al. Whole-Mount immunofluorescence staining, confocal imaging and 3D reconstruction of the sinoatrial and atrioventricular node in the mouse. Journal of Visualized Experiments. (166), 10.3791/62058 (2020). Kumar, P., Si, M., Paulhus, K., Glasscock, E. Microelectrode array recording of sinoatrial node firing rate to identify intrinsic cardiac pacemaking defects in mice. Journal of Visualized Experiments. (173), 10.3791/62735 (2021). Scherschel, K. et al. Location, dissection, and analysis of the murine stellate ganglion. Journal of Visualized Experiments. (166), 10.3791/62026 (2020). Shimura, D., Hunter, J., Katsumata, M., Shaw, R. M. Removal of an internal translational start site from mRNA while retaining expression of the full-length protein. Journal of Visualized Experiments. (181), 10.3791/63405 (2022). Rotzer, R. D. et al. Implantation of combined telemetric ECG and blood pressure transmitters to determine spontaneous baroreflex sensitivity in conscious mice. Journal of Visualized Experiments. (168), 10.3791/62101 (2021). Tomsits, P. et al. Analyzing long-term electrocardiography recordings to detect arrhythmias in mice. Journal of Visualized Experiments. (171), 10.3791/62386 (2021). Grab, M. et al. Development and evaluation of 3D-printed cardiovascular phantoms for interventional planning and training. Journal of Visualized Experiments. (167), 10.3791/62063 (2021).

An optimized method to visualize the goblet cell-associated antigen passages and identify goblet cells in the intestine, conjunctiva, and airway

The goblet cell-associated antigen passages (GAPs), formed in the intestine and conjunctiva, contribute to immune responses by delivering antigens to immune cells. It has been proved that dysfunctions of GAPs may contribute to intestinal inflammation and dry eye disease. GAPs were first discovered in the intestine with the help of intravital two-photon microscopy. The imaging of GAPs was subsequently captured in a frozen section and whole-mount immunofluorescent staining. Both protocols have their features. This study provides a new protocol for visualizing GAPs with better mucus preservation by combining 4% PFA and methanol-Carnoy’s solution (PFA-MC). In addition, embedding samples in paraffin provides an opportunity for antigen retrieval for immunohistochemistry analysis. Using this protocol, we have successfully visualized GAPs in the intestine, intestinal organoids, conjunctiva, and trachea. It is worth mentioning that the visualization of GAPs in intestinal organoids and airways has rarely been discussed before, which may assist researchers in their studies. Moreover, we also discovered the active uptake of dextran of club cells in terminal bronchioles, which may provide a new direction for studying the function of club cells. Overall, we developed a new method to visualize GAP formation and identify GCs simultaneously in the intestine, organoid, conjunctiva, and airway.

Salubrinal Protects Against Cisplatin-Induced Cochlear Hair Cell Endoplasmic Reticulum Stress by Regulating Eukaryotic Translation Initiation Factor 2α Signalling.

ObjectiveCisplatin is a broad-spectrum anti-tumour drug commonly used in clinical practice. However, its ototoxicity greatly limits its clinical application, and no effective method is available to prevent this effect. Endoplasmic reticulum stress (ERS) is reportedly involved in cisplatin ototoxicity, but the exact mechanism remains unclear. Therefore, this study aimed to investigate the role of eukaryotic translation initiation factor 2α (eIF2α) signalling and its dephosphorylation inhibitor salubrinal in cisplatin ototoxicity.MethodsWe evaluated whether salubrinal could protect against cisplatin-induced damage in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and mouse cochlear explants. By knocking down eIF2α, we elucidated the vital role of eIF2α in cisplatin-induced damage in HEI-OC1 cells. Whole-mount immunofluorescent staining and confocal microscopy of mouse cochlear explants and HEI-OC1 cells were performed to analyse cisplatin-induced damage in cochlear hair cells and the auditory cell line.ResultsData suggested salubrinal attenuated cisplatin-induced hair cell injury by inhibiting apoptosis. In addition, salubrinal significantly reduced ERS levels in hair cells via eIF2α signalling, while eIF2α knockdown inhibited the protective effect of salubrinal.SignificanceSalubrinal and eIF2α signalling play a role in protecting against cisplatin-induced ototoxicity, and pharmacological inhibition of eIF2α-mediated ERS is a potential treatment for cisplatin-induced damage in the cochlea and HEI-OC1 cells.

Whole-Mount Immunofluorescence Protocol for 3D Imaging, Reconstruction, and Quantification of Fourth Pharyngeal Arch Artery Formation.

Pharyngeal arch arteries (PAA) are formed early during mouse embryogenesis and remodel soon thereafter into the aortic arch arteries. Failure of these vessels to form or remodel results in congenital heart defects. This protocol is designed to study the formation of the PAA using whole-mount immunofluorescence staining, followed by tissue clearing with benzyl alcohol/benzyl benzoate (BAAB) and imaging by confocal microscopy. The fine cellular resolution obtained with this technique allows the embryonic vasculature of the pharyngeal arch artery endothelium to be visualized by surface rendering and quantitatively analyzed by counting the number of endothelial cells in both the PAA and the vascular plexus surrounding them.

Culture and Imaging of Human Nasal Epithelial Organoids.

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

Bone marrow NLRP3 inflammasome-IL-1β signal regulates post-myocardial infarction megakaryocyte development and platelet production

Platelet plays an important role in the progression of atherosclerosis. Recently it has been reported that myocardial infarction (MI) triggers megakaryopoiesis and thrombopoiesis in the bone marrow and leads to increased circulating platelets, which might contribute to the aggravation of atherosclerosis. However, the underlying mechanisms remain unclear. Here, we analyzed post-MI bone marrow tissue and found that MI induced an upregulation of bone marrow NOD-like Receptor Protein 3 (NLRP3) and subsequent secretion of IL-1β, an essential stimulator of megakaryopoiesis. Targeting NLRP3 using a specific inhibitor MCC950 reduced bone marrow IL-1β expression. Using bone marrow whole-mount immunofluorescence staining combined with flow cytometry, we demonstrated that MCC950 reduced megakaryocyte cellularity and maturity, and effectively attenuated the excessive platelet production after MI. Importantly, mice subjected to MI treated with MCC950 showed a higher survival rate compared with the only MI group. Taken together, this study shows that bone marrow NLRP3-IL-1β signal regulates megakaryocyte development and platelet production after myocardial infarction. It provides a new hint that pharmacological inhibition of NLRP3 might become a potential therapeutic approach for controlling excessive thrombopoiesis after MI.

JAK2V617F Mutant Megakaryocytes Contribute to Hematopoietic Aging in a Murine Model of Myeloproliferative Neoplasm

IntroductionRecent studies implicated megakaryocytes (MKs) in regulating hematopoietic stem cell (HSC) function. Abnormal MK hyperplasia is a hallmark feature of myeloproliferative neoplasms (MPNs). In the present study, we investigated the effects of JAK2V617F-bearing MKs on HSC aging in a murine model of MPN during a 2-yr follow up.MethodsJAK2V617F Flip-Flop (FF1) mice (which carry a Cre-inducible human JAK2V617F gene driven by the human JAK2 promoter) and Pf4-Cre mice (which express Cre under the promoter of platelet factor 4, a MK-specific gene) were crossed to generate MK lineage-specific human JAK2V617F transgenic mouse line (Pf4 +FF1 +).ResultsDuring aging, the Pf4 +FF1 +mice maintained an essential thrombocythemia phenotype with no evidence of transformation to leukemia or myelofibrosis (Figure 1).Myeloid-biased HSCs (Lin -cKit +Sca1 +CD150 +CD48 -CD41 +) were significantly expanded in 1yr old and 2yr old Pf4 +FF1 +mice compared to age-matched control mice, while no difference in myeloid-biased HSC numbers was detected between young (6mo) Pf4 +FF1 +mice and control mice. Competitive repopulation assays and serial transplantation assays showed that HSCs from old Pf4 +FF1 +mice had a reduced engraftment and self-renewal capacity with a skewed differentiation towards the myeloid lineage. Results from the serial transplantation experiments also indicated that the functional decline of HSCs in aged Pf4 +FF1 +mice were HSC-intrinsic and was not reversible. Both cell cycle analysis by Hoechst33342 and Pyronin Y staining and cell proliferation analysis by in vivo BrdU labeling revealed that the JAK2V617F mutant MK niche can promote hematopoietic aging in old Pf4 +FF1 +mice by increasing HSC proliferation/cycling. Taken together, the Pf4 +FF1 +mice demonstrated several hallmarks of accelerated HSC aging.(Figure 2)Next, we examined the hematopoietic microenvironment in the old Pf4 +FF1 + mice. Using whole-mount immunofluorescent staining and confocal imaging, we found that the cKit +HSPCs were located further from MKs in old Pf4 +FF1 +mice compared to old control mice. We also found that CD45 -CD31 +Sca1 - sinusoidal marrow endothelial cell (EC) number (by flow cytometry analysis) and marrow vascular area (by in vivo VE-cadherin staining) were significantly decreased in aged Pf4 +FF1 +mice compared to control mice. Tube formation assays confirmed that conditioned medium from old Pf4 +FF1 +MK culture significantly inhibited EC angiogenesis in vitro. These findings suggest that the JAK2V617F mutant MK niche not only altered its own interaction with HSCs during aging, but also suppressed the vascular niche function to promote HSC aging. (Figure 3)To further understand the mechanisms by which JAK2V617F mutant MKs promote HSC aging, we performed both RNA sequencing and targeted cytokine arrays of wild-type and JAK2V617F mutant MKs from both young (6mo) and old (2yr) Pf4-cre control and Pf4 +FF1 +mice. We found that HSC aging had a profound effect on MK transcriptomic profiles and dysregulated pathways in cell adhesion molecules, MAPK signaling, NF-kappa B signaling, hematopoietic cell lineage, and cytokine-cytokine receptor interaction were highly upregulated in old JAK2V617F mutant MKs compared to young JAK2V617F mutant MKs. FAS ligand, IL-12, tissue inhibitor of metalloproteinases-1, IL-10, IL-6, MIG, macrophage inflammatory protein 1a, GCSF, IL-5, MIP-1g were produced in increased amounts in old mutant MKs compared to old wild-type MKs. Many of these factors are involved in inflammation, angiogenesis, extracellular matrix remodeling, and hematopoiesis regulation.Sensitive PCR assays confirmed that human JAK2 gene was expressed in MKs but not in HSCs in 2yr old Pf4 +FF1 +mice. In vitro co-culture and in vivo co-transplantation assays provided further evidence that the JAK2V617F mutant MKs affected wild-type HSC function directly. Therefore, the hematopoietic aging phenotype we have observed was not caused by any direct effect of the JAK2V617F mutation on HSC function because the Pf4 promoter was ‘leaky’.ConclusionsResults from this study support that, as a hematopoietic niche cell, MKs represent an important connection between the extrinsic and intrinsic mechanisms for HSC aging in MPNs – the JAK2V617F-bearing MKs can alter the hematopoietic niche to accelerate HSC aging, and HSC aging in turn can profoundly remodel the niche e.g. by affecting MK transcriptomics. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Selective Denervation of the Facial Dermato-Muscular Complex in the Rat: Experimental Model and Anatomical Basis.

The facial dermato-muscular system consists of highly specialized muscles tightly adhering to the overlaying skin and thus form a complex morphological conglomerate. This is the anatomical and functional basis for versatile facial expressions, which are essential for human social interaction. The neural innervation of the facial skin and muscles occurs via branches of the trigeminal and facial nerves. These are also the most commonly pathologically affected cranial nerves, often requiring surgical treatment. Hence, experimental models for researching these nerves and their pathologies are highly relevant to study pathophysiology and nerve regeneration. Experimental models for the distinctive investigation of the complex afferent and efferent interplay within facial structures are scarce. In this study, we established a robust surgical model for distinctive exploration of facial structures after complete elimination of afferent or efferent innervation in the rat. Animals were allocated into two groups according to the surgical procedure. In the first group, the facial nerve and in the second all distal cutaneous branches of the trigeminal nerve were transected unilaterally. All animals survived and no higher burden was caused by the procedures. Whisker pad movements were documented with video recordings 4 weeks after surgery and showed successful denervation. Whole-mount immunofluorescent staining of facial muscles was performed to visualize the innervation pattern of the neuromuscular junctions. Comprehensive quantitative analysis revealed large differences in afferent axon counts in the cutaneous branches of the trigeminal nerve. Axon number was the highest in the infraorbital nerve (28,625 ± 2,519), followed by the supraorbital nerve (2,131 ± 413), the mental nerve (3,062 ± 341), and the cutaneous branch of the mylohyoid nerve (343 ± 78). Overall, this surgical model is robust and reliable for distinctive surgical deafferentation or deefferentation of the face. It may be used for investigating cortical plasticity, the neurobiological mechanisms behind various clinically relevant conditions like facial paralysis or trigeminal neuralgia as well as local anesthesia in the face and oral cavity.

The DNA methylation inhibitor RG108 protects against noise-induced hearing loss

BackgroundNoise-induced hearing loss represents a commonly diagnosed type of hearing disability, severely impacting the quality of life of individuals. The current work is aimed at assessing the effects of DNA methylation on noise-induced hearing loss.MethodsBlocking DNA methyltransferase 1 (DNMT1) activity with a selective inhibitor RG108 or silencing DNMT1 with siRNA was used in this study. Auditory brainstem responses were measured at baseline and 2 days after trauma in mice to assess auditory functions. Whole-mount immunofluorescent staining and confocal microcopy of mouse inner ear specimens were performed to analyze noise-induced damage in cochleae and the auditory nerve at 2 days after noise exposure.ResultsThe results showed that noise exposure caused threshold elevation of auditory brainstem responses and cochlear hair cell loss. Whole-mount cochlea staining revealed a reduction in the density of auditory ribbon synapses between inner hair cells and spiral ganglion neurons. Inhibition of DNA methyltransferase activity via a non-nucleoside specific pharmacological inhibitor, RG108, or silencing of DNA methyltransferase-1 with siRNA significantly attenuated ABR threshold elevation, hair cell damage, and the loss of auditory synapses.ConclusionsThis study suggests that inhibition of DNMT1 ameliorates noise-induced hearing loss and indicates that DNMT1 may be a promising therapeutic target.Graphical abstractGraphical Headlights• RG108 protected against noise-induced hearing loss• RG108 administration protected against noise-induced hair cell loss and auditory neural damage.• RG108 administration attenuated oxidative stress-induced DNA damage and subsequent apoptosis-mediated cell loss in the cochlea after noise exposure.

Lymphatic muscle cells contribute to dysfunction of the synovial lymphatic system in inflammatory arthritis in mice

BackgroundOur previous studies reveal that impaired draining function of the synovial lymphatic vessel (LV) contributes to the pathogenesis of inflammatory arthritis, but the cellular and molecular mechanisms involved are not fully understood.ObjectiveTo investigate the involvement of lymphatic muscle cells (LMCs) in mediating impaired LV function in inflammatory arthritis.MethodsTNF transgenic (TNF-Tg) arthritic mice were used. The structure and function of the LVs that drained the hind limbs were examined by whole-mount immunofluorescence staining, electron microscopy, and near-infrared lymphatic imaging. Primary LMCs were treated with TNF, and the changes in proliferation, apoptosis, and functional gene expression were assessed. The roles of the herbal drug, Panax notoginseng saponins (PNS), in arthritis and LVs were studied.ResultsTNF-Tg mice developed ankle arthritis with age, which was associated with abnormalities of LVs: (1) dilated capillary LVs with few branch points, (2) mature LVs with reduced LMC coverage and draining function, and (3) degenerative and apoptotic appearance of LMCs. TNF caused LMC apoptosis, reduced expression of muscle functional genes, and promoted the production of nitric oxide (NO) by lymphatic endothelial cells (LECs). PNS attenuated arthritis, restored LMC coverage and draining function of mature LVs, inhibited TNF-mediated NO expression, and reduced LMC apoptosis.ConclusionThe impaired draining function of LVs in TNF-Tg mice involves LMC apoptosis. TNF promotes LMC death directly and indirectly via NO production by LECs. PNS attenuates arthritis, improves LVs, and prevents TNF-induced LMC apoptosis by inhibiting NO production of LECs. LMCs contribute to the dysfunction of synovial LVs in inflammatory arthritis.