Applying Super-Resolution and Tomography Concepts to Identify Receptive Field Subunits in the Retina.

Spatially nonlinear stimulus integration by retinal ganglion cells lies at the heart of various computations performed by the retina. It arises from the nonlinear transmission of signals that ganglion cells receive from bipolar cells, which thereby constitute functional subunits within a ganglion cell's receptive field. Inferring these subunits from recorded ganglion cell activity promises a new avenue for studying the functional architecture of the retina. This calls for efficient methods, which leave sufficient experimental time to leverage the acquired knowledge for further investigating identified subunits. Here, we combine concepts from super-resolution microscopy and computed tomography and introduce super-resolved tomographic reconstruction (STR) as a technique to efficiently stimulate and locate receptive field subunits. Simulations demonstrate that this approach can reliably identify subunits across a wide range of model variations, and application in recordings of primate parasol ganglion cells validates the experimental feasibility. STR can potentially reveal comprehensive subunit layouts within only a few tens of minutes of recording time, making it ideal for online analysis and closed-loop investigations of receptive field substructure in retina recordings.

Diversity of Ganglion Cell Responses to Saccade-Like Image Shifts in the Primate Retina.

Saccades are a fundamental part of natural vision. They interrupt fixations of the visual gaze and rapidly shift the image that falls onto the retina. These stimulus dynamics can cause activation or suppression of different retinal ganglion cells, but how they affect the encoding of visual information in different types of ganglion cells is largely unknown. Here, we recorded spiking responses to saccade-like shifts of luminance gratings from ganglion cells in isolated marmoset retinas and investigated how the activity depended on the combination of presaccadic and postsaccadic images. All identified cell types, On and Off parasol and midget cells, as well as a type of Large Off cells, displayed distinct response patterns, including particular sensitivity to either the presaccadic or the postsaccadic image or combinations thereof. In addition, Off parasol and Large Off cells, but not On cells, showed pronounced sensitivity to whether the image changed across the transition. Stimulus sensitivity of On cells could be explained based on their responses to step changes in light intensity, whereas Off cells, in particular, parasol and the Large Off cells, seem to be affected by additional interactions that are not triggered during simple light-intensity flashes. Together, our data show that ganglion cells in the primate retina are sensitive to different combinations of presaccadic and postsaccadic visual stimuli. This contributes to the functional diversity of the output signals of the retina and to asymmetries between On and Off pathways and provides evidence of signal processing beyond what is triggered by isolated steps in light intensity.SIGNIFICANCE STATEMENT Sudden eye movements (saccades) shift our direction of gaze, bringing new images in focus on our retinas. To study how retinal neurons deal with these rapid image transitions, we recorded spiking activity from ganglion cells, the output neurons of the retina, in isolated retinas of marmoset monkeys while shifting a projected image in a saccade-like fashion across the retina. We found that the cells do not just respond to the newly fixated image, but that different types of ganglion cells display different sensitivities to the presaccadic and postsaccadic stimulus patterns. Certain Off cells, for example, are sensitive to changes in the image across transitions, which contributes to differences between On and Off information channels and extends the range of encoded stimulus features.

Correction: Samak et al. Dysregulation of Krüppel-like Factor 2 and Myocyte Enhancer Factor 2D Drive Cardiac Microvascular Inflammation and Dysfunction in Diabetes. Int. J. Mol. Sci. 2023, 24, 2482.

In the original publication [...].

The endothelial-enriched lncRNA LINC00607 mediates angiogenic function.

Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNA LINC00607 to be highly enriched in human endothelial cells. LINC00607 was induced in response to hypoxia, arteriosclerosis regression in non-human primates, post-atherosclerotic cultured endothelial cells from patients and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 attenuated VEGF-A-induced angiogenic sprouting. LINC00607 knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression of LINC00607 in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq after LINC00607 knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically, LINC00607 interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout of BRG1 in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion, LINC00607 is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1 to ultimately mediate angiogenesis.

Dysregulation of Krüppel-like Factor 2 and Myocyte Enhancer Factor 2D Drive Cardiac Microvascular Inflammation and Dysfunction in Diabetes.

Cardiovascular complications are the main cause of morbidity and mortality from diabetes. Herein, vascular inflammation is a major pathological manifestation. We previously characterized the cardiac microvascular inflammatory phenotype in diabetic patients and highlighted micro-RNA 92a (miR-92a) as a driver of endothelial dysfunction. In this article, we further dissect the molecular underlying of these findings by addressing anti-inflammatory Krüppel-like factors 2 and 4 (KLF2 and KLF4). We show that KLF2 dysregulation in diabetes correlates with greater monocyte adhesion as well as migratory defects in cardiac microvascular endothelial cells. We also describe, for the first time, a role for myocyte enhancer factor 2D (MEF2D) in cardiac microvascular dysfunction in diabetes. We show that both KLFs 2 and 4, as well as MEF2D, are dysregulated in human and porcine models of diabetes. Furthermore, we prove a direct interaction between miR-92a and all three targets. Altogether, our data strongly qualify miR-92a as a potential therapeutic target for diabetes-associated cardiovascular disease.

Deep Learning-Based Segmentation and Quantification in Experimental Kidney Histopathology.

BACKGROUND: Nephropathologic analyses provide important outcomes-related data in experiments with the animal models that are essential for understanding kidney disease pathophysiology. Precision medicine increases the demand for quantitative, unbiased, reproducible, and efficient histopathologic analyses, which will require novel high-throughput tools. A deep learning technique, the convolutional neural network, is increasingly applied in pathology because of its high performance in tasks like histology segmentation. METHODS: We investigated use of a convolutional neural network architecture for accurate segmentation of periodic acid-Schiff-stained kidney tissue from healthy mice and five murine disease models and from other species used in preclinical research. We trained the convolutional neural network to segment six major renal structures: glomerular tuft, glomerulus including Bowman's capsule, tubules, arteries, arterial lumina, and veins. To achieve high accuracy, we performed a large number of expert-based annotations, 72,722 in total. RESULTS: Multiclass segmentation performance was very high in all disease models. The convolutional neural network allowed high-throughput and large-scale, quantitative and comparative analyses of various models. In disease models, computational feature extraction revealed interstitial expansion, tubular dilation and atrophy, and glomerular size variability. Validation showed a high correlation of findings with current standard morphometric analysis. The convolutional neural network also showed high performance in other species used in research-including rats, pigs, bears, and marmosets-as well as in humans, providing a translational bridge between preclinical and clinical studies. CONCLUSIONS: We developed a deep learning algorithm for accurate multiclass segmentation of digital whole-slide images of periodic acid-Schiff-stained kidneys from various species and renal disease models. This enables reproducible quantitative histopathologic analyses in preclinical models that also might be applicable to clinical studies.

"Empowering" Cardiac Cells via Stem Cell Derived Mitochondrial Transplantation- Does Age Matter?

With cardiovascular diseases affecting millions of patients, new treatment strategies are urgently needed. The use of stem cell based approaches has been investigated during the last decades and promising effects have been achieved. However, the beneficial effect of stem cells has been found to being partly due to paracrine functions by alterations of their microenvironment and so an interesting field of research, the "stem- less" approaches has emerged over the last years using or altering the microenvironment, for example, via deletion of senescent cells, application of micro RNAs or by modifying the cellular energy metabolism via targeting mitochondria. Using autologous muscle-derived mitochondria for transplantations into the affected tissues has resulted in promising reports of improvements of cardiac functions in vitro and in vivo. However, since the targeted treatment group represents mainly elderly or otherwise sick patients, it is unclear whether and to what extent autologous mitochondria would exert their beneficial effects in these cases. Stem cells might represent better sources for mitochondria and could enhance the effect of mitochondrial transplantations. Therefore in this review we aim to provide an overview on aging effects of stem cells and mitochondria which might be important for mitochondrial transplantation and to give an overview on the current state in this field together with considerations worthwhile for further investigations.

The aging common marmoset's immune system: From junior to senior.

The social, health, and economic challenges of a steadily increasing aging population demand the use of appropriate translational animal models to address questions like healthy aging, vaccination strategies, or potential interventions during the aging process. Due to their genetic proximity to humans, especially nonhuman primates (NHPs) with a relatively short generation period compared to humans, qualify as excellent animal models for these purposes. The use of common marmosets (Callithrix jacchus) in gerontology research steadily increased over the last decades, yet important information about their aging parameters are still missing. We therefore aimed to characterize their aging immune system by comprehensive flow cytometric phenotyping of blood immune cells from juvenile, adult, aging, and geriatric animals. Aged and geriatric animals displayed clear signs of immunosenescence. A decline in CD4/CD8 ratio, increased expression of HLA-DR and PD-1, higher frequencies of CD95+ memory cells, alterations in cytokine secretion, and a decline in the proliferative capacity proved T cell senescence in aging marmosets. Also, the B cell compartment was affected by age-related changes: while overall B cell numbers remained stable with advancing age, expression of the activation marker CD80 increased and immunoglobulin M expression decreased. Interestingly, marmoset B cell memory subset distribution rather mirrored the human situation than that of other NHP. CD21+ CD27- naïve B cell frequencies decreased while those of CD21- CD27- tissue memory B cells increased with age. Furthermore, frequencies and numbers of NK cells as part of the innate immune system declined with advancing age. Thus, the observed immunological changes in common marmosets over their life span revealed several similarities to age-related changes in humans and encourages further studies to strengthen the common marmoset as a potential aging model.

Blood pressure as prognostic marker for body condition, cardiovascular, and metabolic diseases in the common marmoset (Callithrix jacchus).

BACKGROUND: The increasing life span of Callithrix jacchus in combination with the occurrence of metabolic and age-dependent diseases requires improved health surveillance for this species. METHODS: The health status of 56 marmosets was studied using a non-invasive blood pressure (BP) device. Age-, weight-, and sex-dependent changes were analyzed. Four animals with striking BP findings had follow-up exams. RESULTS: Physiological and pathological BP values could be defined. BP positively correlated with age and weight, while no effect of sex could be found. Measurement time for female and older animals was shorter than for male and younger individuals. Further analysis of the suspicious patients revealed renal or hepatic diseases and cardiac alterations. CONCLUSION: The description of age and weight influences on BP delivers physiological and pathological values for common marmosets. This may contribute to the understanding of aging process and cardiology in this primate species.

The influence of gluten on clinical and immunological status of common marmosets (Callithrix jacchus).

BACKGROUND: Common marmosets (Callithrix jacchus) are susceptible to gastrointestinal diseases. Sensitivity to nutritional elements, for example gluten, has been suggested, but a serological screening has not been performed yet. METHODS: A gluten-containing diet was offered to 24 animals, followed by a gluten-free diet. During these diets, serum IgA antibodies to gliadin (AGA), tissue transglutaminase (tTG), deamidated gliadin (ADGA), and glycoprotein 2 (AGP2A) were determined. Body weight, diarrhea, and other clinical symptoms were recorded. RESULTS: Gluten increased AGA, tTG, and AGP2A concentrations in 13 of 24 animals. A significant decline of AGA and AGP2A was seen on gluten withdrawal. Positive (AGA, tTG) animals presented diarrhea more frequently on gluten-containing diet and showed significantly increased body weight on gluten-free diet compared to negative animals. CONCLUSION: Gluten ingestion caused gastrointestinal symptoms in common marmosets, which disappeared on gluten withdrawal. Considering the immunological response to both diets, gluten sensitivity seems to be most likely.

Functional Cardiovascular Characterization of the Common Marmoset (Callithrix jacchus).

Appropriate cardiovascular animal models are urgently needed to investigate genetic, molecular, and therapeutic approaches, yet the translation of results from the currently used species is difficult due to their genetic distance as well as their anatomical or physiological differences. Animal species that are closer to the human situation might help to bridge this translational gap. The common marmoset (Callithrix jacchus) is an interesting candidate to investigate certain heart diseases and cardiovascular comorbidities, yet a basic functional characterization of its hemodynamic system is still missing. Therefore, cardiac functional analyses were performed by utilizing the invasive intracardiac pressure-volume loops (PV loop) system in seven animals, magnetic resonance imaging (MRI) in six animals, and echocardiography in five young adult male common marmosets. For a direct comparison between the three methods, only data from animals for which all three datasets could be acquired were selected. All three modalities were suitable for characterizing cardiac function, though with some systemic variations. In addition, vena cava occlusions were performed to investigate the load-independent parameters collected with the PV loop system, which allowed for a deeper analysis of the cardiac function and for a more sensitive detection of the alterations in a disease state, such as heart failure or certain cardiovascular comorbidities.

Evaluation of MR-safe bioptomes for MR-guided endomyocardial biopsy in minipigs: a potential radiation-free clinical approach.

BACKGROUND: Diagnostic accuracy of endomyocardial biopsy could improve if clinically safe magnetic resonance (MR)-compatible bioptomes were available. We explored two novel MR-compatible cardiac bioptomes for performance, safety, and clinical viability, employing in vivo minipig trials and phase-contrast synchrotron radiation computed microtomography (SRµCT). METHODS: Analysis of ex vivo obtained pig endomyocardial biopsies was performed using phase-contrast SRµCT and conventional two-dimensional histology. The technical performance was evaluated by measuring volume, inner and outer integrities, compression, and histological diagnostic value in 3 sets (6 per set) of biopsies for each experimental bioptome. The bioptomes were tested in vivo in 3 healthy minipigs per bioptome. The clinical feasibility was evaluated by procedural and cutting success as well as histological diagnostic value. RESULTS: The bioptome with the 'grind-grind' design achieved similar values to control in compression (p = 0.822), inner (p = 0.628), and outer (p = 0.507), integrities ex vivo. It showed a better performance in the in vivo real-time MRI setting demonstrating a higher cutting success (91.7%) than the 'grind-anvil' (86.2%) design. In both ex vivo and in vivo evaluations, the 'grind-grind' design displayed sufficient diagnostic value (83% and 95%). The 'grind-anvil' design showed adequate diagnostic value both ex vivo and in vivo (78% and 87.5%) but was not comparable to control according to the three-dimensional (3D) analysis. CONCLUSION: A novel MR-compatible bioptome was identified as plausible in a clinical setting. Additionally, SRµCT and subsequent 3D structural analysis could be valuable in the label-free investigation of myocardial tissue at a micrometer level. RELEVANCE STATEMENT: Implementation of MR-guided biopsy can improve animal studies on structural myocardial changes at any point in an experimental setup. With further improvements in guiding catheters, MR-guided biopsy, using the new bioptome, has a potential to increase quality and diagnostic accuracy in patients both with structural and inflammatory cardiomyopathies. KEY POINTS: • Novel MR-compatible bioptomes show promise for a clinical application. • SRµCT enabled detailed analysis of endomyocardial biopsies. • The bioptomes showed adequate in vivo performance without major complications.

No evidence for age-related alterations in the marmoset retina.

The physiological aging process of the retina is accompanied by various and sometimes extensive changes: Macular degeneration, retinopathies and glaucoma are the most common findings in the elderly and can potentially lead to irreversible visual disablements up to blindness. To study the aging process and to identify possible therapeutic targets to counteract these diseases, the use of appropriate animal models is mandatory. Besides the most commonly used rodent species, a non-human primate, the common marmoset (Callithrix jacchus) emerged as a promising animal model of human aging over the last years. However, the visual aging process in this species is only partially characterized, especially with regard to retinal aberrations. Therefore, we assessed here for the first time potential changes in retinal morphology of the common marmoset of different age groups. By cell type specific immunolabeling, we analyzed different cell types and distributions, potential photoreceptor and ganglion cell loss, and structural reorganization. We detected no signs of age-related differences in staining patterns or densities of various cell populations. For example, there were no signs of photoreceptor degeneration, and there was only minimal sprouting of rod bipolar cells in aged retinas. Altogether, we describe here the maintenance of a stable neuronal architecture, distribution and number of different cell populations with only mild aberrations during the aging process in the common marmoset retina. These findings are in stark contrast to previously reported findings in rodent species and humans and deserve further investigations to identify the underlying mechanisms and possible therapeutic targets.

Developmental errors in the common marmoset retina.

Although retinal organization is remarkably conserved, morphological anomalies can be found to different extents and varieties across animal species with each presenting unique characteristics and patterns of displaced and misplaced neurons. One of the most widely used non-human primates in research, the common marmoset (Callithrix jaccus) could potentially also be of interest for visual research, but is unfortunately not well characterized in this regard. Therefore, the aim of our study was to provide a first time description of structural retinal layering including morphological differences and distinctive features in this species. Retinas from animals (n = 26) of both sexes and different ages were immunostained with cell specific antibodies to label a variety of bipolar, amacrine and ganglion cells. Misplaced ganglion cells with somata in the outermost part of the inner nuclear layer and rod bipolar cells with axon terminals projecting into the outer plexiform layer instead of the inner plexiform layer independent of age or sex of the animals were the most obvious findings, whereas misplaced amacrine cells and misplaced cone bipolar axon terminals occurred to a lesser extent. With this first time description of developmental retinal errors over a wide age range, we provide a basic characterization of the retinal system of the common marmosets, which can be taken into account for future studies in this and other animal species. The finding of misplaced ganglion cells and misplaced bipolar cell axon terminals was not reported before and displays an anatomic variation worthwhile for future analyzes of their physiological and functional impact.

Label-free imaging of age-related cardiac structural changes in non-human primates using multiphoton nonlinear microscopy.

Heart failure is one of the most common causes of morbidity and mortality. Both maturational abnormalities and age-associated cardiac pathologies contribute to heart failure. Imaging-based assessment to discern detailed cardiac structure at various maturational stages is imperative for understanding mechanisms behind cardiac growth and aging. Using multiphoton nonlinear optical microscopy (NLOM) based label-free imaging, we investigated cardiac structural composition in a human-relevant aging model, the common marmoset monkey (Callithrix jacchus). Animals were divided into three different age groups including neonatal, young adult and old. By devising a unique strategy for segregating collagen and myosin emitted second harmonic generation (SHG) signals, we performed a volumetric assessment of collagen and total scattering tissue (collagen + myosin). Aged marmoset hearts exhibited an increase in collagen and total scattering tissue volume at the sites of severe tissue remodelling indicating age-related cardiac fibrosis. Significantly low scattering tissue volume in neonatal marmoset hearts was attributed to a lack of binding between the myofibrils in maturing cardiac tissue. Comprehensive quantitative assessment of structural composition during maturation and aging of marmoset hearts revealed significant differences in myofibril length, alignment, curvature and angular distribution. In conclusion, label-free high-resolution NLOM facilitates visualization and quantification of subcellular structural features for understanding vital age-related morphological alterations in the marmoset heart.

Cardiac MRI in common marmosets revealing age-dependency of cardiac function.

The aim of this study was to establish a feasible and robust magnetic resonance imaging protocol for the quantitative assessment of cardiac function in marmosets and to present normal values of cardiac function across different ages from young adult, middle-aged, to very old clinically healthy animals. Cardiac MRI of 33 anesthetized marmosets at the age of 2-15 years was performed at 9.4 T using IntraGate-FLASH that operates without any ECG-triggering and breath holding. Normalized to post-mortem heart weight, the left ventricular end-diastolic volume (LV-EDV) was significantly reduced in older marmosets. The LV end-systolic volume (LV-ESV) and the LV stroke volume (LV-SV) showed a similar trend while the LV ejection fraction (LV-EF) and wall thickening remained unchanged. Similar observations were made for the right ventricle. Moreover, the total ventricular myocardial volume was lower in older monkeys while no significant difference in heart weight was found. In conclusion, IntraGate-FLASH allowed for quantification of left ventricular cardiac function but seems to underestimate the volumes of the right ventricle. Although less strong and without significant sex differences, the observed age related changes were similar to previously reported findings in humans supporting marmosets as a model system for age related cardiovascular human diseases.

Revisiting a quarter of a century of simian immunodeficiency virus (SIV)-associated cardiovascular diseases at the German Primate Center.

Human immunodeficiency virus (HIV) comorbidities have become clinically more important due to antiretroviral therapy. Although therapy increases life expectancy, it does not completely suppress immune activation and its associated complications. The simian immunodeficiency virus (SIV)-infected rhesus macaque (Macaca mulatta) represents a valuable model for the investigation of SIV-associated diseases. Although cardiovascular (CV) changes are common in HIV-infected patients, there are only a few reports on the incidence of CV findings in SIV-infected animals. In addition, potential associations between pathohistological findings and hematological parameters are still unclear. We therefore conducted a retrospective analysis of 195 SIV-infected rhesus macaques that were euthanized with AIDS-related symptoms at the German Primate Center, Goettingen, over a 25-year period. Pathological findings were correlated with hematological data. The main findings included myocarditis (12.8 %), endocarditis (9.7 %), and arteriopathy (10.3 %) in various organs. Thrombocytopenia occurred more frequently in macaques with endocarditis or arteriopathy than in macaques without CV disease (80 % in animals with endocarditis, 60 % in animals with arteriopathy, p < 0.0001 and p = 0.0016 , respectively). Further investigations of the interaction between coagulation markers, proinflammatory cytokines, and biomarkers associated with endothelial dysfunction (e.g., D-dimers) and histological data (vascular wall structure) may unravel the mechanisms underlying HIV/SIV-associated CV comorbidities.