Whole-mount Immunofluorescence Research Articles

Improved whole-mount immunofluorescence protocol for consistent and robust labeling of adult Drosophila melanogaster adipose tissue.

Energy storage and endocrine functions of the Drosophila fat body make it an excellent model for elucidating mechanisms that underlie physiological and pathophysiological organismal metabolism. Combined with Drosophila's robust genetic and immunofluorescence microscopy toolkits, studies of Drosophila fat body function are ripe for cell biological analysis. Unlike the larval fat body, which is easily removed as a single, cohesive sheet of tissue, isolating intact adult fat body proves to be more challenging, thus hindering consistent immunofluorescence labeling even within a single piece of adipose tissue. Here, we describe an improved approach to handling Drosophila abdomens that ensures full access of the adult fat body to solutions generally used in immunofluorescence labeling protocols. In addition, we assess the quality of fluorescence reporter expression and antibody immunoreactivity in response to variations in fixative type, fixation incubation time, and detergent used for cellular permeabilization. Overall, we provide several recommendations for steps in a whole mount staining protocol that results in consistent and robust immunofluorescence labeling of the adult Drosophila fat body.

P-693 Three-dimensional modeling of follicular microenvironment dynamics in the human adult ovary

Abstract Study question How does the ovarian microenvironment, involving neural fibers, vascular networks, and immune cells, interplay with follicular dynamics in the human adult ovary? Summary answer Neural fibers growing into the ovarian cortex and vascular network expansion were associated with primordial follicle activation and growth. Macrophages associated with antral follicular atresia. What is known already The ovarian follicles, responsible for preserving and supplying oocytes, play a crucial role in fertility, with their fate influenced by the ovarian microenvironment. However, the three-dimensional (3D), irregular, and dynamic structural changes of the ovarian microenvironment are difficult to interpret using two-dimensional analysis. 3D-imaging technologies have been developed to reveal intricate structural and functional relationships particularly in rodents, but these approaches have not been widely adopted for human ovarian research. Study design, size, duration Our study aims to explore the follicular microenvironment dynamics in the human adult ovary. In this work, we optimized whole-mount immunofluorescence using TUBB3, PODXL, PECAM1, ACTA2 and CD68, and conducted 3D imaging on intact adult ovarian tissue, offering a visualization of the 3D structures and dynamic changes within ovarian follicles, neural fibers, vascular networks, and immune cells in human adult ovary at the single-cell level. Participants/materials, setting, methods The human ovarian samples were collected from donors (N = 5) who underwent oophorectomy after testosterone treatment. Part of the intact ovary, including cortex and medulla, was fixed and used for whole-mount immunofluorescence using markers for neural fibers (TUBB3), blood vessels (PODXL, PECAM1, ACTA2), and immune cells (CD68). Subsequently, an enhanced iDiSCO method was employed for tissue clearance. Finally, 3D images were acquired using a lightsheet microscope, followed by image analysis and 3D reconstruction using Imaris software. Main results and the role of chance Our results showed that TUBB3-labeled nerve fibers predominantly distributed in the ovarian cortex, with few presented in the medullary region. Notably, their presence surrounding secondary and antral follicles was limited. We observed a rich distribution of PODXL-labeled capillaries surrounding primordial and primary follicles in the cortical layer of the adult ovary. As follicles progressed to the secondary stage, PECAM1-labeled vasculature contributed to the construction of a vascular network enveloping the secondary follicles and connecting extensively to larger-caliber arteries containing (ACTA2+) smooth muscle cells. In antral follicles, large larger-caliber arteries were also detected, maintaining proximity to the theca cell layer. Interestingly, in cases of follicular atresia, vascular invasion within the follicular cavity was observed. Additionally, clusters of CD68+ macrophages exhibited a scattered distribution around the follicles at various stages, with a significant accumulation in atretic small-antral follicles. Limitations, reasons for caution Our study was conducted on ovaries from a limited number of donors after gender-affirming surgery. Although we aimed to provide insights into the ovarian microenvironment, we were so far unable to obtain ovaries with large antral follicles, ovulatory follicles and the formation of the corpus luteum. Wider implications of the findings The views provided by 3D-image analysis of the human ovary will augment the current knowledge of the complex interactions between the follicles and the ovarian microenvironment. 3D ovarian mapping will also provide a roadmap for generating oocytes in vitro for fertility preservation and ovarian disease modelling. Trial registration number N/A.

Protocol for the Growth and Maturation of hiPSC-Derived Kidney Organoids using Mechanically Defined Hydrogels.

With recent advances in the reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs), gene editing technologies, and protocols for the directed differentiation of stem cells into heterogeneous tissues, iPSC-derived kidney organoids have emerged as a useful means to study processes of renal development and disease. Considerable advances guided by knowledge of fundamental renal developmental signaling pathways have been made with the use of exogenous morphogens to generate more robust kidney-like tissues in vitro. However, both biochemical and biophysical microenvironmental cues are major influences on tissue development and self-organization. In the context of engineering the biophysical aspects of the microenvironment, the use of hydrogel extracellular scaffolds for organoid studies has been gaining interest. Two families of hydrogels have recently been the subject of significant attention: self-assembling peptide hydrogels (SAPHs), which are fully synthetic and chemically defined, and gelatin methacryloyl (GelMA) hydrogels, which are semi-synthetic. Both can be used as support matrices for growing kidney organoids. Based on our recently published work, we highlight methods describing the generation of human iPSC (hiPSC)-derived kidney organoids and their maturation within SAPHs and GelMA hydrogels. We also detail protocols required for the characterization of such organoids using immunofluorescence imaging. Together, these protocols should enable the user to grow hiPSC-derived kidney organoids within hydrogels of this kind and evaluate the effects that the biophysical microenvironment provided by the hydrogels has on kidney organoid maturation. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Directed differentiation of human induced pluripotent stem cells (hiPSCs) into kidney organoids and maturation within mechanically tunable self-assembling peptide hydrogels (SAPHs) Alternate Protocol: Encapsulation of day 9 nephron progenitor aggregates in gelatin methacryloyl (GelMA) hydrogels. Support Protocol 1: Human induced pluripotent stem cell (hiPSC) culture. Support Protocol 2: Organoid fixation with paraformaldehyde (PFA) Basic Protocol 2: Whole-mount immunofluorescence imaging of kidney organoids. Basic Protocol 3: Immunofluorescence of organoid cryosections.

Protocol for the Growth and Maturation of hiPSC-Derived Kidney Organoids using Mechanically Defined Hydrogels.

With recent advances in the reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs), gene editing technologies, and protocols for the directed differentiation of stem cells into heterogeneous tissues, iPSC-derived kidney organoids have emerged as a useful means to study processes of renal development and disease. Considerable advances guided by knowledge of fundamental renal developmental signaling pathways have been made with the use of exogenous morphogens to generate more robust kidney-like tissues in vitro. However, both biochemical and biophysical microenvironmental cues are major influences on tissue development and self-organization. In the context of engineering the biophysical aspects of the microenvironment, the use of hydrogel extracellular scaffolds for organoid studies has been gaining interest. Two families of hydrogels have recently been the subject of significant attention: self-assembling peptide hydrogels (SAPHs), which are fully synthetic and chemically defined, and gelatin methacryloyl (GelMA) hydrogels, which are semi-synthetic. Both can be used as support matrices for growing kidney organoids. Based on our recently published work, we highlight methods describing the generation of human iPSC (hiPSC)-derived kidney organoids and their maturation within SAPHs and GelMA hydrogels. We also detail protocols required for the characterization of such organoids using immunofluorescence imaging. Together, these protocols should enable the user to grow hiPSC-derived kidney organoids within hydrogels of this kind and evaluate the effects that the biophysical microenvironment provided by the hydrogels has on kidney organoid maturation. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Directed differentiation of human induced pluripotent stem cells (hiPSCs) into kidney organoids and maturation within mechanically tunable self-assembling peptide hydrogels (SAPHs) Alternate Protocol: Encapsulation of day 9 nephron progenitor aggregates in gelatin methacryloyl (GelMA) hydrogels. Support Protocol 1: Human induced pluripotent stem cell (hiPSC) culture. Support Protocol 2: Organoid fixation with paraformaldehyde (PFA) Basic Protocol 2: Whole-mount immunofluorescence imaging of kidney organoids. Basic Protocol 3: Immunofluorescence of organoid cryosections.

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.

Smurf1 Modulates Smad Signaling Pathway in Fibrotic Cataract Formation.

TGF-β/BMP signaling pathway plays a significant role in fibrotic cataract. Smurf1, a ubiquitin protein ligase, regulates the TGF-β/BMP signaling pathway through the ubiquitin-proteasome system (UPS). This study aims to investigate the role of Smurf1 in the progression of fibrotic cataract and its underlying mechanism. We used a mouse model of injury-induced anterior subcapsular cataract (ASC) and administered the Smurf1 inhibitor A01 for in vivo investigations. RNA sequencing was performed to examine global gene expression changes. Protein levels were assessed by Simple Western analysis. The volume of subcapsular opacity was determined using whole-mount immunofluorescence of lens anterior capsules. Lentivirus was utilized to establish cell lines with Smurf1 knockdown or overexpression in SRA01/04. Lens epithelial cell (LEC) proliferation was evaluated by CCK8 and EdU assays. Cell cycle profile was determined by flow cytometry. LEC migration was measured using Transwell and wound healing assays. The mRNA levels of genes associated with cell proliferation, migration, epithelial-mesenchymal transition (EMT), TGF-β/BMP pathway, and UPS were upregulated in mouse ASC model. Smurf1 mRNA and protein levels were upregulated in lens capsules of patients and mice with ASC. Anterior chamber injection of A01 inhibited ASC formation and EMT. In vitro, Smurf1 knockdown reduced proliferation, migration and TGF-β2-induced EMT of LECs, concomitant with the upregulation of Smad1, Smad5, and pSmad1/5. Conversely, overexpression of Smurf1 showed opposite phenotypes. Smurf1 regulates fibrotic cataract progression by influencing LEC proliferation, migration, and EMT through the modulation of the Smad signaling pathway, offering a novel target for the fibrotic cataract treatment.

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.

Inactivation of ERK1/2 signaling mediates dysfunction of basal meningeal lymphatic vessels in experimental subdural hematoma.

Rationale: Meningeal lymphatic vessels (MLVs) are essential for the clearance of subdural hematoma (SDH). However, SDH impairs their drainage function, and the pathogenesis remains unclear. Herein, we aimed to understand the pathological mechanisms of MLV dysfunction following SDH and to test whether atorvastatin, an effective drug for SDH clearance, improves meningeal lymphatic drainage (MLD). Methods: We induced SDH models in rats by injecting autologous blood into the subdural space and evaluated MLD using Gadopentetate D, Evans blue, and CFSE-labeled erythrocytes. Whole-mount immunofluorescence and transmission electron microscopy were utilized to detect the morphology of MLVs. Phosphoproteomics, western blot, flow cytometry, and in vitro experiments were performed to investigate the molecular mechanisms underlying dysfunctional MLVs. Results: The basal MLVs were detected to have abundant valves and play an important role in draining subdural substances. Following SDH, these basal MLVs exhibited disrupted endothelial junctions and dilated lumen, leading to impaired MLD. Subsequent proteomics analysis of the meninges detected numerous dephosphorylated proteins, primarily enriched in the adherens junction, including significant dephosphorylation of ERK1/2 within the meningeal lymphatic endothelial cells (LECs). Subdural injection of the ERK1/2 kinase inhibitor PD98059 resulted in dilated basal MLVs and impaired MLD, resembling the dysfunctional MLVs observed in SDH. Moreover, inhibiting ERK1/2 signaling severely disrupted intercellular junctions between cultured LECs. Finally, atorvastatin was revealed to protect the structure of basal MLVs and accelerate MLD following SDH. However, these beneficial effects of atorvastatin were abolished when combined with PD98059. Conclusion: Our findings demonstrate that SDH induces ERK1/2 dephosphorylation in meningeal LECs, leading to disrupted basal MLVs and impaired MLD. Additionally, we reveal a beneficial effect of atorvastatin in improving MLD.

Cxcl1 monomer-dimer equilibrium controls neutrophil extravasation.

The chemokine Cxcl1 plays a crucial role in recruiting neutrophils in response to infection. The early events in chemokine-mediated neutrophil extravasation involve a sequence of highly orchestrated steps including rolling, adhesion, arrest, and diapedesis. Cxcl1 function is determined by its properties of reversible monomer-dimer equilibrium and binding to Cxcr2 and glycosaminoglycans. Here, we characterized how these properties orchestrate extravasation using intravital microscopy of the cremaster. Compared to WT Cxcl1, which exists as both a monomer and a dimer, the trapped dimer caused faster rolling, less adhesion, and less extravasation. Whole-mount immunofluorescence of the cremaster and arrest assays confirmed these data. Moreover, the Cxcl1 dimer showed impaired LFA-1-mediated neutrophil arrest that could be attributed to impaired Cxcr2-mediated ERK signaling. We conclude that Cxcl1 monomer-dimer equilibrium and potent Cxcr2 activity of the monomer together coordinate the early events in neutrophil recruitment.

Spatial Mapping Reveals Distinct Erythroid Niches in Mice and Humans during Development and Stress

Like hematopoiesis, erythropoiesis requires erythroid niche nurturing and support. While extensive studies have revealed various components and functions of the HSC niches, the erythroid niche is less characterized. The most studied erythroid niche is the bone marrow erythroblastic island (EBI), which comprises a central macrophage surrounded by developing erythroblasts. However, most of the reported studies of EBI involve in vitro reconstitution of mixed cell populations that do not recapitulate in vivo niches. Here, we utilized multiple spatial mapping technologies, including spatial transcriptomics and whole-mount imaging studies, to characterize erythropoiesis in mouse and human hematopoietic tissues during development and under stress. We first utilized the Visium platform from 10x Genomics to study the spatial transcriptomic profiles of the E14.5 mouse fetal liver, which is the major organ for murine fetal definitive erythropoiesis. The Visium platform contains barcoded spots with a diameter of 55 µm, which is suboptimal for single-cell analyses but ideal for the investigation of cell-cell interactions within their microenvironment. We found that each hepatic lobe exhibited different cluster distributions, indicating distinct cell compositions among different anatomic loci. Further deconvolution of the clusters and co-analysis with corresponding single-cell RNA sequencing data revealed that there was a higher positive correlation between erythroid cells and C1q+ macrophages compared to other macrophages, suggesting that C1q+ macrophages are more likely to be EBI macrophages in mouse. C1q+ macrophages exhibited high expression of known genes involved in EBI macrophage function and erythropoiesis. Cyclic immunofluorescence (CyCIF) and whole-mount immunofluorescence analyses further demonstrated the co-localization of C1q+ macrophages with erythroid cells in the E14.5 fetal liver. This strong positive correlation between C1q+ macrophages and erythroid cells was also observed across different stages of fetal liver development, newborn bone marrow, and adult spleen under physiologic and stress conditions. These findings demonstrate that C1q+ macrophages as EBI macrophages are conserved across different hematopoietic organs in mice. We next applied the same technologies to human hematopoietic tissues during development and stress. In contrast to the mouse, we did not observe a strong positive correlation between erythroid cells and C1q+ macrophages or other macrophages in the human fetal liver. Instead, there is a strong association between erythroid progenitor and maturing erythroid cells. This strong association is also found in different developmental stages of the human fetal liver as well as the bone marrow, which becomes even stronger in regenerating human bone marrow after chemotherapy. We also recapitulated these findings in a human bone marrow organoid model. These results demonstrate the erythroid cell self-sustaining capacities in human erythropoiesis. Taken together, our findings reveal the distinct erythroid niches in mice and humans during development and stress, which provides a comprehensive understanding of erythropoiesis during evolution.

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.

Disruption of Col9a2 expression leads to defects in osteochondral homeostasis and osteoarthritis-like phenotype in mice

Background/ObjectiveAs one of the branched chains of Type IX collagen (Col9), Collagen IX alpha2 (Col9a2) has been reported to be associated with several orthopedic conditions. However, the relationship between Col9a2 and knee osteoarthritis (KOA) remains to be elucidated. MethodsTo probe the relationship between Col9a2 and KOA, we performed a systematic analysis of Col9a2-deficient (Col9a2−/−) mice using whole-mount skeletal staining, Micro-CT (μCT), biomechanics, histomorphometry, immunohistochemistry (IHC), immunofluorescence (IF) and Elisa. ResultsWe found that the subchondral bone (SCB) in the knee joint of Col9a2−/− mice became sparse and deformed in the early stage, with altered bone morphometric parameters, reduced load-bearing capacity, dysfunctional bone homeostasis (decreased osteogenesis capacity and elevated bone resorption capacity), diminished cartilage proteoglycans and disrupted cartilage extracellular matrix (ECM) anabolism and catabolism compared with the Col9a2+/+ mice. In the late stage, the cartilage degeneration in Col9a2−/− mice were particularly pronounced compared to Col9a2+/+ mice, as evidenced by severe cartilage destruction and a marked reduction in cartilage thickness and area. ConclusionOverall, Col9a2 is essential for maintaining osteochondral homeostasis in the knee joint of mice, and the absence of this gene is accompanied by distinct sclerosis of the SCB and a reduction in load-bearing capacity; in the late stage, in the lack of SCB stress inhibition, excessive load is consistently exerted on the cartilage, ultimately leading to osteoarthritic-like articular cartilage damage.

Disruption of BMP4 signaling is associated with laryngeal birth defects in a mouse model

Laryngeal birth defects are considered rare, but they can be life-threatening conditions. The BMP4 gene plays an important role in organ development and tissue remodeling throughout life. Here we examined its role in laryngeal development complementing similar efforts for the lung, pharynx, and cranial base. Our goal was to determine how different imaging techniques contribute to a better understanding of the embryonic anatomy of the normal and diseased larynx in small specimens. Contrast-enhanced micro CT images of embryonic larynx tissue from a mouse model with Bmp4 deletion informed by histology and whole-mount immunofluorescence were used to reconstruct the laryngeal cartilaginous framework in three dimensions. Laryngeal defects included laryngeal cleft, laryngeal asymmetry, ankylosis and atresia. Results implicate BMP4 in laryngeal development and show that the 3D reconstruction of laryngeal elements provides a powerful approach to visualize laryngeal defects and thereby overcoming shortcomings of 2D histological sectioning and whole mount immunofluorescence.

BMP-4 and BMP-7 Inhibit EMT in a Model of Anterior Subcapsular Cataract in Part by Regulating the Notch Signaling Pathway.

The proliferation, migration, and epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) are believed to be the pathological mechanisms underlying anterior subcapsular cataract (ASC). Bone morphogenetic proteins (BMPs) inhibit transforming growth factor-beta (TGF-β)-induced fibrosis in the lens. Herein, we aimed to further clarify the roles of BMP-4/BMP-7 in the progression and the underlying mechanisms of fibrotic cataract. BMP-4/BMP-7, TGF-β2, jagged-1 peptide, or DAPT were applied in a mouse injury-induced ASC model and in the human LEC cell line SRA01/04. The volume of opacity was examined by a slit lamp and determined by lens anterior capsule whole-mount immunofluorescence. Global gene expression changes were assessed by RNA sequencing, and the levels of individual mRNAs were validated by real-time PCR. Protein expression was determined by the Simple Western sample dilution buffer. Cell proliferation was examined by CCK8 and EdU assays, and cell migration was measured by Transwell and wound healing assays. Anterior chamber injection of BMP-4/BMP-7 significantly suppressed subcapsular opacification formation. RNA sequencing of the mouse ASC model identified the Notch pathway as a potential mechanism involved in BMP-mediated inhibition of ASC. Consistently, BMP-4/BMP-7 selectively suppressed Notch1 and Notch3 and their downstream genes, including Hes and Hey. BMP-4/BMP-7 or DAPT suppressed cell proliferation by inducing G1 cell cycle arrest. BMP-4/BMP-7 also inhibited TGF-β2-induced cell migration and EMT by modulating the Notch pathway. BMP-4/BMP-7 attenuated ASC by inhibiting proliferation, migration, and EMT of LECs via modulation of the Notch pathway, thereby providing a new avenue for ASC treatment.

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).

Brain endothelial CXCL12 attracts protective natural killer cells during ischemic stroke

BackgroundThe innate lymphoid cell (ILC) family consists of NK cells, ILC type 1, 2, 3 and lymphoid tissue inducer cells. They have been shown to play important roles in homeostasis and immune responses and are generally considered tissue resident. Not much is known about the presence of ILC members within the central nervous system and whether they are tissue resident in this organ too. Therefore, we studied the presence of all ILC members within the central nervous system and after ischemic brain insult.MethodsWe used the photothrombotic ischemic lesion method to induce ischemic lesions within the mouse brain. Using whole-mount immunofluorescence imaging, we established that the ILCs were present at the rim of the lesion. We quantified the increase of all ILC members at different time-points after the ischemic lesion induction by flow cytometry. Their migration route via chemokine CXCL12 was studied by using different genetic mouse models, in which we induced deletion of Cxcl12 within the blood–brain barrier endothelium, or its receptor, Cxcr4, in the ILCs. The functional role of the ILCs was subsequently established using the beam-walk sensorimotor test.ResultsHere, we report that ILCs are not resident within the mouse brain parenchyma during steady-state conditions, but are attracted towards the ischemic stroke. Specifically, we identify NK cells, ILC1s, ILC2s and ILC3s within the lesion, the highest influx being observed for NK cells and ILC1s. We further show that CXCL12 expressed at the blood–brain barrier is essential for NK cells and NKp46+ ILC3s to migrate toward the lesion. Complementary, Cxcr4-deficiency in NK cells prevents NK cells from entering the infarct area. Lack of NK cell migration results in a higher neurological deficit in the beam-walk sensorimotor test.ConclusionsThis study establishes the lack of ILCs in the mouse central nervous system at steady-state and their migration towards an ischemic brain lesion. Our data show a role for blood–brain barrier-derived CXCL12 in attracting protective NK cells to ischemic brain lesions and identifies a new CXCL12/CXCR4-mediated component of the innate immune response to stroke.

Amniotic Membrane Scaffolds Support Organized Muscle Regeneration in A Murine Volumetric Muscle Defect Model.

Background:Current treatment for volumetric muscle loss is limited to muscle transfer or acellular collagen scaffold (ACS) therapies that are associated with donor site morbidity and nonfunctional fibrosis, respectively. The aim of this study is to assess the utility of amniotic membrane scaffold (AMS) for volumetric muscle loss treatment.Methods:Murine quadriceps defects were created and randomized to three groups (n = 5/group): untreated controls, ACS, and AMS. In vivo muscle regeneration volume was quantified by MRI and microcomputed tomography. Muscle explants were analyzed using standard histology and whole-mount immunofluorescence at 8 weeks.Results:The cross-sectional muscle regeneration ratio was 0.64 ± 0.3 for AMS, 0.48 ± 0.07 for ACS, and 0.4 0 ± 0.03 for controls as assessed by MRI (P = 0.09) and 0.61 ± 0.28 for AMS, 0.50 ± 0.06 for ACS, and 0.43 ± 0.04 for controls as assessed by microcomputed tomography (P = 0.2). Histologically, AMS demonstrated significantly higher cellular density (900 ± 2 70 nuclei/high powered field) than ACS (210 ± 36) and control (130 ± 4) groups (P = 0.05). Immunofluorescence for laminin (AMS 623 ± 11 versus ACS 339 ± 3 versus control 115 ± 7; P < 0.01) and myosin heavy chain (AMS 509 ± 7 versus ACS 288 ± 5 versus control 84 ± 5; P = 0.03) indicated greater organized muscle fiber formation with AMS.Conclusion:AMS mediated muscle healing was characterized by increased cellular infiltration and organized muscle formation when compared with controls and ACS.