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Summary: To address the needs of the life sciences community and the pharmaceutical industry in pre-clinical drug development to both maintain and continuously assess tissue metabolism and function with simple and rapid systems, we improved on the initial BaroFuse to develop it into a fully functional, pumpless, scalable multi-channel fluidics instrument that continuously measures changes in oxygen consumption and other endpoints in response to test compounds. We and several other laboratories assessed it with a wide range of tissue types including retina, pancreatic islets, liver, and hypothalamus with both aqueous and gaseous test compounds. The setup time was less than an hour for all collaborating groups, and there was close agreement between data obtained from the different laboratories. This easy-to-use system reliably generates real-time metabolic and functional data from tissue and cells in response to test compounds that will address a critical need in basic and applied research. Motivation: There is a critical need for developing in vitro methods that provide mechanistic insight at the tissue and intracellular levels as well as to reduce the need for in vivo studies. Flow culture is recognized as superior to static for both maintenance and assessment of tissue, as static systems are unable to maintain viability of most tissue preparations that are not cellular monolayers. However, technical challenges and a need for increased amounts of biological samples are impediments to their routine use in the majority of laboratories in the life sciences. We have addressed these two issues by developing a simple-to-use flow system that utilizes gas pressure instead of peristaltic or syringe pumps to drive multi-channel microflow. The delivery of steady flow over a wide range of flow rates enables small amounts of tissue to be maintained and assessed.

Summary: Nutritional deprivation occurring in most preterm infants postnatally can induce hyperglycemia, a significant and independent risk factor for suppressing physiological retinal vascularization (Phase I retinopathy of prematurity (ROP)), leading to compensatory but pathological neovascularization. Amino acid supplementation reduces retinal neovascularization in mice. Little is known about amino acid contribution to Phase I ROP. In mice modeling hyperglycemia-associated Phase I ROP, we found significant changes in retinal amino acids (including most decreased L-leucine, L-isoleucine, and L-valine). Parenteral L-isoleucine suppressed physiological retinal vascularization. In premature infants, severe ROP was associated with a higher mean intake of parenteral versus enteral amino acids in the first two weeks of life after adjustment for treatment group, gestational age at birth, birth weight, and sex. The number of days with parenteral amino acids support independently predicted severe ROP. Further understanding and modulating amino acids may help improve nutritional intervention and prevent Phase I ROP.

Abstract Background Microglia cells represent the resident innate immune cells of the retina and are important for retinal development and tissue homeostasis. However, dysfunctional microglia can have a negative impact on the structural and functional integrity of the retina under native and pathological conditions. Methods In this study, we examined interferon-regulatory factor 8 (Irf8)–deficient mice to determine the transcriptional profile, morphology, and temporospatial distribution of microglia lacking Irf8 and to explore the effects on retinal development, tissue homeostasis, and formation of choroidal neovascularisation (CNV). Results Our study shows that Irf8-deficient MG exhibit a considerable loss of microglial signature genes accompanied by a severely altered MG morphology. An in-depth characterisation by fundus photography, fluorescein angiography, optical coherence tomography and electroretinography revealed no major retinal abnormalities during steady state. However, in the laser-induced CNV model, Irf8-deficient microglia showed an increased activity of biological processes critical for inflammation and cell adhesion and a reduced MG cell density near the lesions, which was associated with significantly increased CNV lesion size. Conclusions Our results suggest that loss of Irf8 in microglia has negligible effects on retinal homeostasis in the steady state. However, under pathological conditions, Irf8 is crucial for the transformation of resident microglia into a reactive phenotype and thus for the suppression of retinal inflammation and CNV formation.

PurposeTo decipher the transcriptional signature of macrophages of the human vitreous, also known as hyalocytes, and compare it to the profiles of other myeloid cell populations including human blood-derived monocytes, macrophages, and brain microglia.MethodsThis study involves a total of 13 patients of advanced age with disorders of the vitreoretinal interface undergoing vitrectomy at the University Eye Hospital Freiburg between 2018 and 2019. Vitreal hyalocytes were analyzed by fluorescence-activated cell sorting (FACS) and isolated as CD45+CD11b+CX3CR1+Mat-Mac+ cells using a FACS-based sorting protocol. RNA extraction, library preparation and RNA sequencing were performed and the sequencing data was analyzed using the Galaxy web platform. The transcriptome of human hyalocytes was compared to the transcriptional profile of human blood-derived monocytes, macrophages and brain microglia obtained from public databases. Protein validation for selected factors was performed by immunohistochemistry on paraffin sections from three human donor eyes.ResultsOn average, 383 ± 233 hyalocytes were isolated per patient, resulting in 128 pg/μl ± 76 pg/μl total RNA per sample. RNA sequencing revealed that SPP1, FTL, CD74, and HLA-DRA are among the most abundantly expressed genes in hyalocytes, which was confirmed by immunofluorescence for CD74, FTL, and HLA-DRA. Gene ontology (GO) enrichment analysis showed that biological processes such as “humoral immune response,” “leukocyte migration,” and “antigen processing and presentation of peptide antigen” (adjusted p < 0.001) are dominating in vitreal hyalocytes. While the comparison of the gene expression profiles of hyalocytes and other myeloid cell populations showed an overall strong similarity (R2 > 0.637, p < 0.001), hyalocytes demonstrated significant differences with respect to common leukocyte-associated factors. In particular, transcripts involved in the immune privilege of the eye, such as POMC, CD46, and CD86, were significantly increased in hyalocytes compared to other myeloid cell subsets.ConclusionHuman hyalocytes represent a unique and distinct innate immune cell population specialized and adapted for the tissue-specific needs in the human vitreous. Vitreal hyalocytes are characterized by a strong expression of genes related to antigen processing and presentation as well as immune modulation. Thus, hyalocytes may represent an underestimated mediator in vitreoretinal disease and for the immune privilege of the eye.