Multi-organ immunity on a 3D-printed multi-tissue chip with tubing-free impeller pump.

Multi-organ-on-chip systems (MOOCs) have the potential to mimic communication between organ systems and reveal mechanisms of health and disease. However, many existing MOOCs are challenging for non-experts to implement, due to complex tubing, electronics, or pump mechanisms. In addition, few MOOCs have incorporated immune organs such as the lymph node (LN), limiting their applicability to critical events such as vaccination. Here we developed a 3D-printed, user-friendly device and companion tubing-free impeller pump to co-culture two or more tissue samples, including a LN, under a recirculating common media. Native tissue structure and immune function were incorporated by maintaining slices of murine LN tissue ex vivo in 3D- printed mesh supports for at least 24 hr. In a two-compartment model of a LN and an upstream injection site, vaccination of the multi-tissue chip was similar to in vivo vaccination in terms of locations of antigen accumulation and acute changes in activation markers and gene expression in the LN. We anticipate that in the future, this flexible platform will enable models of multi-organ immune responses throughout the body.

Spatially resolved quantification of oxygen consumption rate in ex vivo lymph node slices.

Cellular metabolism has been closely linked to activation state in cells of the immune system, and the oxygen consumption rate (OCR) in particular serves as a valuable metric for assessing metabolic activity. Several oxygen sensing assays have been reported for cells in standard culture conditions. However, none have provided a spatially resolved, optical measurement of local oxygen consumption in intact tissue samples, making it challenging to understand regional dynamics of consumption. Therefore, here we established a system to monitor the rates of oxygen consumption in ex vivo tissue slices, using murine lymphoid tissue as a case study. By integrating an optical oxygen sensor into a sealed perfusion chamber and incorporating appropriate correction for photobleaching of the sensor and of tissue autofluorescence, we were able to visualize and quantify rates of oxygen consumption in tissue. This method revealed for the first time that the rate of oxygen consumption in naïve lymphoid tissue was higher in the T cell region compared to the B cell and cortical regions. To validate the method, we measured OCR in the T cell regions of naïve lymph node slices using the optical assay and estimated the consumption rate per cell. The predictions from the optical assay were similar to reported values and were not significantly different from those of the Seahorse metabolic assay, a gold standard method for measuring OCR in cell suspensions. Finally, we used this method to quantify the rate of onset of tissue hypoxia for lymph node slices cultured in a sealed chamber and showed that continuous perfusion was sufficient to maintain oxygenation. In summary, this work establishes a method to monitor oxygen consumption with regional resolution in intact tissue explants, suitable for future use to compare tissue culture conditions and responses to stimulation.

Spatially resolved quantification of oxygen consumption rate in ex vivo lymph node slices.

Cellular metabolism has been closely linked to activation state in cells of the immune system, and the oxygen consumption rate (OCR) in particular serves as a valuable metric for assessing metabolic activity. Several oxygen sensing assays have been reported for cells in standard culture conditions. However, none have provided a spatially resolved, optical measurement of local oxygen consumption in intact tissue samples, making it challenging to understand regional dynamics of consumption. Therefore, here we established a system to monitor the rates of oxygen consumption in ex vivo tissue slices, using murine lymphoid tissue as a case study. By integrating an optical oxygen sensor into a sealed perfusion chamber and incorporating appropriate correction for photobleaching of the sensor and of tissue autofluorescence, we were able to visualize and quantify rates of oxygen consumption in tissue. This method revealed for the first time that the rate of oxygen consumption in naïve lymphoid tissue was higher in the T cell region compared to the B cell and cortical regions. To validate the method, we measured OCR in the T cell regions of naïve lymph node slices using the optical assay and estimated the consumption rate per cell. The predictions from the optical assay were similar to reported values and were not significantly different from those of the Seahorse metabolic assay, a gold standard method for measuring OCR in cell suspensions. Finally, we used this method to quantify the rate of onset of tissue hypoxia for lymph node slices cultured in a sealed chamber and showed that continuous perfusion was sufficient to maintain oxygenation. In summary, this work establishes a method to monitor oxygen consumption with regional resolution in intact tissue explants, suitable for future use to compare tissue culture conditions and responses to stimulation.

A Rare Association of Hypomagnesemia and Posterior Reversible Encephalopathy Syndrome (PRES).

Posterior reversible encephalopathy syndrome (PRES) is a rare neurologic disorder that presents with variable symptoms and symmetrical abnormal white matter signaling most commonly of the occipital and parietal lobes on magnetic resonance imaging (MRI). PRES, also known as reversible posterior leukoencephalopathy syndrome (RPLS) is commonly associated with hypertension. Hypomagnesemia's association with PRES has been rarely reported. Here, we report a patient with severe hypomagnesemia that presented with PRES syndrome that improved with magnesium replacement. Hypomagnesemia should be considered an underlying etiology in patients presenting with PRES syndrome and should be promptly treated. The presentation can often be concerning for acute cerebrovascular accidents with symptoms of dysarthria and upper motor neuron symptoms, such as facial droop, dysarthria, and gait instability. Differential diagnosis of PRES often includes rostral brainstem infarction, transient ischemic attack, infectious encephalopathy, and metabolic/toxic encephalopathy, which is evaluated in the description of the case. The most common presentation of RPLS/PRES includes altered mental status, drowsiness, seizure, vomiting, alterations in speech including dysarthria, and visual disturbance. The first signs noted are commonly lethargy and somnolence. In this case, the patient presented notably with initial symptoms of dysarthria of speech and facial droop, with serum hypomagnesemia in which symptoms corrected rapidly with the administration of intravenous magnesium sulfate.

Anatomical location of injected microglia in different activation states and time course of injury determines survival of retinal ganglion cells after optic nerve crush.

Background: Activated microglia release harmful substances to retinal ganglion cells (RGCs), but may also benefit by removing cellular debris and secreting neurotrophic factors. These paradoxical roles remain controversial because the nature and time-course of the injury that defines their role is unknown. The aim of this study was to determine if pharmacological manipulation of microglia to acquire a pro-inflammatory or pro-survival phenotype will exacerbate or enhance neuronal survival after injury.Material and methods: Treated HAP I (highly aggressively proliferating immortalized) microglia were injected into the vitreous or tail vein (T V) of female Sprague-Dawley rats. Retinas were examined at 4-14 days following optic nerve crush (ONC) and the number of surviving RGCs was determined.Results: Injection of untreated HAP I cells resulted in the greater loss of RGCs early after ONC when injected into the vitreous and later after ONC when injected into the T V. LP S activated HAP I cells injected into the vitreous resulted in greater RGC loss with and without injury. When injected into the T V with ONC there was no loss of RGCs 4 days after ONC but greater loss afterwards. Minocycline treated HAP I cells injected into the vitreous resulted in greater RGC survival than untreated HAP I cells. However, when injected into the T V with ONC there was greater loss of RGCs. These results suggest that optic nerve signals attract extrinsic microglia to the retina, resulting in a proinflammatory response.Conclusion: Neuroprotection or cytotoxicity of microglia depends on the type of activation, time course of the injury, and if they act on the axon or cell body.

We show here that neuroprotection is not solely determined by the microglial activation state but factors such as the environment and time-course of the injury.Culture microglia can be treated in vitro and then injected in vivo.The cells migrate to the site of injury, cell body of retinal ganglion cells if in the vitreous or to the optic nerve if injected in the tail vein.Retinal ganglion cell death is dependent on the location the microglia act, time-course of injury, and activation state.Proinflammatory microglia can be neuroprotective early in the injury when the primary site of action is on the axons whereas hypoactivated microglia are neuroprotective early in injury when they act on the soma. Later in the injury, both become detrimental.

Rapid Fabrication by Digital Light Processing 3D Printing of a SlipChip with Movable Ports for Local Delivery to Ex Vivo Organ Cultures.

SlipChips are two-part microfluidic devices that can be reconfigured to change fluidic pathways for a wide range of functions, including tissue stimulation. Currently, fabrication of these devices at the prototype stage requires a skilled microfluidic technician, e.g., for wet etching or alignment steps. In most cases, SlipChip functionality requires an optically clear, smooth, and flat surface that is fluorophilic and hydrophobic. Here, we tested digital light processing (DLP) 3D printing, which is rapid, reproducible, and easily shared, as a solution for fabrication of SlipChips at the prototype stage. As a case study, we sought to fabricate a SlipChip intended for local delivery to live tissue slices through a movable microfluidic port. The device was comprised of two multi-layer components: an enclosed channel with a delivery port and a culture chamber for tissue slices with a permeable support. Once the design was optimized, we demonstrated its function by locally delivering a chemical probe to slices of hydrogel and to living tissue with up to 120 µm spatial resolution. By establishing the design principles for 3D printing of SlipChip devices, this work will enhance the ability to rapidly prototype such devices at mid-scale levels of production.

Progressive Loss of Retinal Ganglion Cells in Activating Protein-2ß Neural Crest Cell Knockout Mice.

Purpose: Our lab has shown that conditionally disrupting the transcription factor activating protein 2ß (Tfap2b) gene, responsible for the activating protein-2ß (AP-2ß) transcription factor, exclusively in cranial neural crest cells (AP-2ß NCC KO), leads to anterior segment dysgenesis and a closed angle phenotype. The purpose of the current study is to determine if there is a progressive loss of retinal ganglion cells (RGCs) in the mutant over time and whether this loss was associated with macroglial activity changes and elevated intraocular pressure (IOP).Methods: Using the Cre-loxP system, we generated a conditional knockout of Tfap2b exclusively in cranial NCC (AP-2ß NCC KO). Immunohistochemistry was performed using anti-Brn3a, anti-GFAP and anti-Vimentin antibodies. IOP was measured using a tonometer and the data was analyzed using GraphPad Prism software. Brn3a and DAPI positive cells were counted using Image-J and statistical analysis was performed with GraphPad Prism software.Results: Our findings revealed that while no statistical difference in Brn3a expression was observed between wild-type and mutant mice at postnatal day (P) 4 or P10, at P40 (p < .01) and P42 (p < .0001) Brn3a expression was significantly reduced in the mutant retina at the region of the ONH. There was also increased expression of glial fibrillary acidic protein (GFAP) by Müller cells in the AP-2ß NCC KO mice at P35 and P40, indicating the presence of neuroinflammation. Moreover, increased IOP was observed starting at P35 and continuing at P40 and P42 (p < .0001 for all three ages examined).Conclusions: Together, these findings suggest that the retinal damage observed in the KO mouse becomes apparent by P40 after increased IOP was observed at P35 and progressed over time. The AP-2ß NCC KO mouse may therefore be a novel experimental model for glaucoma.

Acute Lymph Node Slices Are a Functional Model System to Study Immunity Ex Vivo.

The lymph node is a highly organized and dynamic structure that is critical for facilitating the intercellular interactions that constitute adaptive immunity. Most ex vivo studies of the lymph node begin by reducing it to a cell suspension, thus losing the spatial organization, or fixing it, thus losing the ability to make repeated measurements. Live murine lymph node tissue slices offer the potential to retain spatial complexity and dynamic accessibility, but their viability, level of immune activation, and retention of antigen-specific functions have not been validated. Here we systematically characterized live murine lymph node slices as a platform to study immunity. Live lymph node slices maintained the expected spatial organization and cell populations while reflecting the 3D spatial complexity of the organ. Slices collected under optimized conditions were comparable to cell suspensions in terms of both 24-h viability and inflammation. Slices responded to T cell receptor cross-linking with increased surface marker expression and cytokine secretion, in some cases more strongly than matched lymphocyte cultures. Furthermore, slices processed protein antigens, and slices from vaccinated animals responded to ex vivo challenge with antigen-specific cytokine secretion. In summary, lymph node slices provide a versatile platform to investigate immune functions in spatially organized tissue, enabling well-defined stimulation, time-course analysis, and parallel read-outs.

Detergent wash improves vaccinated lymph node handling ex vivo.

Lymph nodes (LNs) are essential secondary immune organs where the adaptive immune response is generated against most infections and vaccines. We recently described the use of live ex vivo LN slices to study the dynamics of adaptive immunity. However, when working with reactive lymph nodes from vaccinated animals, the tissues frequently became dislodged from the supportive agarose matrix during slicing, leading to damage that prevented downstream analysis. Because reactive lymph nodes expand into the surrounding adipose tissue, we hypothesized that dislodging was a result of excess lipids on the collagen capsule of the LN, and that a brief wash with a mild detergent would improve LN interaction with the agarose without damaging tissue viability or function. Therefore, we tested the use of digitonin on improving slicing of vaccinated LNs. Prior to embedding, LNs were quickly dipped into a digitonin solution and washed in saline. Lipid droplets were visibly removed by this procedure. A digitonin wash step prior to slicing significantly reduced the loss of LN during slicing from 13 to 75% to 0-25%, without substantial impact on viability. Capture of fluorescent microparticles, uptake and processing of protein antigen, and cytokine secretion in response to a vaccine adjuvant, R848, were all unaffected by the detergent wash. This novel approach will enable ex vivo analysis of the generation of adaptive immune response in LNs in response to vaccinations and other immunotherapies.

AP-2ß is required for formation of the murine trabecular meshwork and Schlemm's canal.

Previously, we have shown that Tfap2b, the gene encoding transcription factor AP-2ß, is needed for normal mouse eye development. Specifically, targeted loss of Tfap2b in neural crest cells (NCCs)1 and their derivatives, particularly the periocular mesenchyme (POM), resulted in anterior segment defects affecting the cornea and angle tissue. These defects were further associated with an increase in intraocular pressure (IOP). The present study investigates the underlying changes in embryonic and postnatal POM cell development and differentiation caused by loss of AP-2ß in the NCCs, particularly in the structures that control aqueous outflow, using Wnt1Cre+/-; Tfap2b-/lox; tdTomatolox/+ mice (AP-2ß neural crest cell knockout or AP-2ß NCC KO). Toluidine blue-stained sections and ultrathin sections stained with uranyl acetate and lead citrate were used to assess morphology and ultrastructure, respectively. Immunohistochemistry of KO and control eyes was performed at embryonic day (E) 15.5, E18.5, postnatal day (P) 1, P7 and P14 using phospho-histone H3 (PH3), α-smooth muscle actin (α-SMA), myocilin and endomucin antibodies, as well as a TUNEL assay. Conditional deletion of AP-2ß in the NCC-derived POM resulted in defects that appeared during both embryogenesis and postnatal stages. Fate mapping of the knockout cells in the mutants revealed that the POM migrated appropriately into the eye during embryogenesis. However, during postnatal stages a significant reduction in POM proliferation in the angle region was observed in the mutants compared to controls. This was accompanied by a lack of expression of appropriate trabecular meshwork and Schlemm's canal markers. This is the first study to show that AP-2ß is required for development and differentiation of the trabecular meshwork and Schlemm's canal. Together, these defects likely contributed to the elevated intraocular pressure (IOP) previously reported in the AP-2ß NCC KO mice.

Labelling primary immune cells using bright blue fluorescent nanoparticles.

Tracking cell movements is an important aspect of many biological studies. Reagents for cell tracking must not alter the biological state of the cell and must be bright enough to be visualized above background autofluorescence, a particular concern when imaging in tissue. Currently there are few reagents compatible with standard UV excitation filter sets (e.g. DAPI) that fulfill those requirements, despite the development of many dyes optimized for violet excitation (405 nm). A family of boron-based fluorescent dyes, difluoroboron ß-diketonates, has previously served as bio-imaging reagents with UV excitation, offering high quantum yields and wide excitation peaks. In this study, we investigated the use of one such dye as a potential cell tracking reagent. A library of difluoroboron dibenzoylmethane (BF2dbm) conjugates were synthesized with biocompatible polymers including: poly(l-lactic acid) (PLLA), poly(ε-caprolactone) (PCL), and block copolymers with poly(ethylene glycol) (PEG). Dye-polymer conjugates were fabricated into nanoparticles, which were stable for a week at 37 °C in water and cell culture media, but quickly aggregated in saline. Nanoparticles were used to label primary splenocytes; phagocytic cell types were more effectively labelled. Labelling with nanoparticles did not affect cellular viability, nor basic immune responses. Labelled cells were more easily distinguished when imaged on a live tissue background than those labelled with a commercially available UV-excitable cytoplasmic labelling reagent. The high efficiency in terms of both fluorescence and cellular labelling may allow these nanoparticles to act as a short-term cell labelling strategy while wide excitation peaks offer utility across imaging and analysis platforms.

Re-description of Chinese mitten crab Eriocheir sinensis H. Milne Edwards, 1853 (Crustacea: Brachyura: Grapsoidea: Varunidae) zoeal development using confocal laser scanning microscopy.

A number of authors have described the zoeal development of the Chinese mitten crab, Eriocheir sinensis H. Milne Edwards, 1853, while some of these studies only recognised 5 zoeal stages, two of them described six. The present study re-examined the zoeal stages of E. sinensis from laboratory-reared material using confocal laser scanning microscopy and visualised the images using the open-source software programmes ImageJ and Drishti. From these images 6 zoeal stages were re-described and compared with all previous larval descriptions of the Chinese mitten crab. Comments on the variation of some setal characters are also described in zoeal stages IV-VI.

Microbial-tubeworm associations in a 440 million year old hydrothermal vent community.

Microorganisms are the chief primary producers within present-day deep-sea hydrothermal vent ecosystems, and play a fundamental role in shaping the ecology of these environments. However, very little is known about the microbes that occurred within, and structured, ancient vent communities. The evolutionary history, diversity and the nature of interactions between ancient vent microorganisms and hydrothermal vent animals are largely undetermined. The oldest known hydrothermal vent community that includes metazoans is preserved within the Ordovician to early Silurian Yaman Kasy massive sulfide deposit, Ural Mountains, Russia. This deposit contains two types of tube fossil attributed to annelid worms. A re-examination of these fossils using a range of microscopy, chemical analysis and nano-tomography techniques reveals the preservation of filamentous microorganisms intimately associated with the tubes. The microfossils bear a strong resemblance to modern hydrothermal vent microbial filaments, including those preserved within the mineralized tubes of the extant vent polychaete genus Alvinella The Yaman Kasy fossil filaments represent the oldest animal-microbial associations preserved within an ancient hydrothermal vent environment. They allude to a diverse microbial community, and also demonstrate that remarkable fine-scale microbial preservation can also be observed in ancient vent deposits, suggesting the possible existence of similar exceptionally preserved microfossils in even older vent environments.

Conditional Deletion of AP-2α and AP-2ß in the Developing Murine Retina Leads to Altered Amacrine Cell Mosaics and Disrupted Visual Function.

Purpose: The combined action of the activating protein-2 (AP-2) transcription factors, AP-2α and AP-2ß, is important in early retinal development, specifically in the formation of horizontal cells. However, in previous studies, it was not possible to analyze postnatal development and function of additional retinal subtypes. Methods: We used a double conditional deletion of AP-2α and AP-2ß from the retina to further examine the combinatory role of these genes in retinal cell patterning and function in postnatal adult mice as measured by Voronoi domain area and nearest-neighbor distance spatial analyses and ERGs, respectively. Results: Conditional deletion of both AP-2α and AP-2ß from the retina resulted in a variety of abnormalities, including the absence of horizontal cells, defects in the photoreceptor ribbons in which synapses failed to form, along with evidence of aberrant amacrine cell arrangement. Although no significant changes in amacrine cell population numbers were observed in the double mutants, significant irregularities in the mosaic patterning of amacrine cells was observed as demonstrated by both Voronoi domain areas and nearest-neighbor distances analyses. These changes were further accompanied by an alteration in the retinal response to light as recorded by ERGs. In particular, in the double-mutant mice lacking AP-2α and AP-2ß, the b-wave amplitude, representative of interneuron signal processing, was significantly reduced compared with control littermates. Conclusions: Together these findings demonstrate the requirement for both AP-2α and AP-2ß in proper amacrine mosaic patterning and a normal functional light response in the retina.

Qualitative Assessment of Clerkship Students' Perspectives of the Topics of Pain and Addiction in their Preclinical Curriculum.

OBJECTIVE: A majority of physicians feel poorly trained in the treatment of chronic pain and addiction. As such, it is critical that medical students receive appropriate education in both pain management and addiction. The purpose of this study was to assess the pre-clinical curriculum in pain medicine and addiction from the perspective of students after they had completed their pre-clinical training and to assess what they perceived as the strengths and weaknesses of their training. METHODS: The authors conducted focused interviews among clinical medical students who had completed at least 6 months of clerkships. The interviews targeted the students' retrospective opinions about the pre-clinical curriculum and their preparedness for clinical encounters with either pain or addiction-related issues during their rotations. Coders thematically analyzed the de-identified interview transcripts, with consensus reached through discussion and code modification. RESULTS: Themes that emerged through the focused interviews included: fragmented curricular structure (and insufficient time) for pain and addiction medicine, not enough specific treatment strategies for pain or addiction, especially for complex clinical scenarios, and lack of a trained work-force to provide guidance in the management of pain and addiction. CONCLUSION: This study demonstrated the feasibility of gathering student perspectives to inform changes to improve the pre-clinical curriculum in pain and addiction medicine. Students identified multiple areas for improvement at the pre-clerkship level, which have informed updates to the curriculum. More research is needed to determine if curricular changes based on student feedback lead to improved learning outcomes.

The Use of Laser Microirradiation to Investigate the Roles of Cohesins in DNA Repair.

In addition to their mitotic and transcriptional functions, cohesin plays critical roles in DNA damage response (DDR) and repair. Specifically, cohesin promotes homologous recombination (HR) repair of DNA double-strand breaks (DSBs), which is conserved from yeast to humans, and is a critical effector of ATM/ATR DDR kinase-mediated checkpoint control in mammalian cells. Optical laser microirradiation has been instrumental in revealing the damage site-specific functions of cohesin and, more recently, uncovering the unique role of cohesin-SA2, one of the two cohesin complexes uniquely present in higher eukaryotes, in DNA repair in human cells. In this review, we briefly describe what we know about cohesin function and regulation in response to DNA damage, and discuss the optimized laser microirradiation conditions used to analyze cohesin responses to DNA damage in vivo.

Conditional deletion of AP-2ß in mouse cranial neural crest results in anterior segment dysgenesis and early-onset glaucoma.

Anterior segment dysgenesis (ASD) encompasses a group of developmental disorders in which a closed angle phenotype in the anterior chamber of the eye can occur and 50% of patients develop glaucoma. Many ASDs are thought to involve an inappropriate patterning and migration of the periocular mesenchyme (POM), which is derived from cranial neural crest cells (NCCs) and mesoderm. Although, the mechanism of this disruption is not well understood, a number of transcriptional regulatory molecules have previously been implicated in ASDs. Here, we investigate the function of the transcription factor AP-2ß, encoded by Tfap2b, which is expressed in NCCs and their derivatives. Wnt1-Cre-mediated conditional deletion of Tfap2b in NCCs resulted in post-natal ocular defects typified by opacity. Histological data revealed that the conditional AP-2ß NCC knockout (KO) mutants exhibited dysgenesis of multiple structures in the anterior segment of the eye including defects in the corneal endothelium, corneal stroma, ciliary body and disruption in the iridocorneal angle with adherence of the iris to the cornea. We further show that this phenotype leads to a significant increase in intraocular pressure and a subsequent loss of retinal ganglion cells and optic nerve degeneration, features indicative of glaucoma. Overall, our findings demonstrate that AP-2ß is required in the POM for normal development of the anterior segment of the eye and that the AP-2ß NCC KO mice might serve as a new and exciting model of ASD and glaucoma that is fully penetrant and with early post-natal onset.

Single-nucleus RNA-seq of differentiating human myoblasts reveals the extent of fate heterogeneity.

Myoblasts are precursor skeletal muscle cells that differentiate into fused, multinucleated myotubes. Current single-cell microfluidic methods are not optimized for capturing very large, multinucleated cells such as myotubes. To circumvent the problem, we performed single-nucleus transcriptome analysis. Using immortalized human myoblasts, we performed RNA-seq analysis of single cells (scRNA-seq) and single nuclei (snRNA-seq) and found them comparable, with a distinct enrichment for long non-coding RNAs (lncRNAs) in snRNA-seq. We then compared snRNA-seq of myoblasts before and after differentiation. We observed the presence of mononucleated cells (MNCs) that remained unfused and analyzed separately from multi-nucleated myotubes. We found that while the transcriptome profiles of myoblast and myotube nuclei are relatively homogeneous, MNC nuclei exhibited significant heterogeneity, with the majority of them adopting a distinct mesenchymal state. Primary transcripts for microRNAs (miRNAs) that participate in skeletal muscle differentiation were among the most differentially expressed lncRNAs, which we validated using NanoString. Our study demonstrates that snRNA-seq provides reliable transcriptome quantification for cells that are otherwise not amenable to current single-cell platforms. Our results further indicate that snRNA-seq has unique advantage in capturing nucleus-enriched lncRNAs and miRNA precursors that are useful in mapping and monitoring differential miRNA expression during cellular differentiation.