Immunofluorescent imaging is an indispensable technique to study morphology and molecular aspects in tissues. Classical approaches make it necessary to cut physical sections of tissue samples to ...overcome the limited penetration depth of light, restricting the available information to two dimensions. Recent advances in tissue-clearing techniques enable imaging of fluorescently labeled organs and entire organisms on a cellular level in three dimensions without the need of sectioning. Volume imaging of immunolabeled and cleared tissues started a new era of systems biology, because these techniques provide information on connectivity and circuits, especially in structures with projections in three dimensions such as vascular or nervous systems. The variety of published clearing protocols allows the imaging of every organ with a single exception: the eye. Whole-eye clearing approaches were unsuccessful so far due to the strong pigmentation of the retinal pigment epithelium. Here, we present a new protocol that combines a highly effective melanin bleaching step with solvent-based clearing, termed EyeCi. The protocol is compatible with immunolabeling as demonstrated by the visualization of ocular and retinal vasculature in the intact mouse eye by means of light-sheet fluorescence microscopy. This novel protocol is rapid (1 week) and inexpensive, hence allowing high-throughput, high resolution analysis of vascular architecture of healthy and diseased eyes, in its native, three-dimensional organization within intact eyeballs. Volume imaging of whole cleared eyeballs further enables three-dimensional surface reconstruction and automated quantification of choroidal and retinal vasculature extending ocular imaging to a global level. Thus, EyeCi represents an extension to state-of-the-art light microscopy techniques and is potentially suitable for the investigation of vascular leakage or neovascularization processes.
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•EyeCi enables immunolabeling and clearing of pigmented mouse eyes.•CD31 immunofluorescence labeling imaged by light-sheet fluorescence microscopy.•Characteristic blood vessel structures could be identified in cleared mouse eyes.•Automated quantification of retinal capillaries.•Choroidal and retinal vasculature in 3D organization within intact eyeballs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Oxidative stress and hypoxia in the retinal pigment epithelium (RPE) have long been considered major risk factors in the pathophysiology of age-related macular degeneration (AMD), but systematic ...investigation of the interplay between these two risk factors was lacking. For this purpose, we treated a human RPE cell line (ARPE-19) with sodium iodate (SI), an oxidative stress agent, together with dimethyloxalylglycine (DMOG) which leads to stabilization of hypoxia-inducible factors (HIFs), key regulators of cellular adaptation to hypoxic conditions. We found that HIF stabilization aggravated oxidative stress-induced cell death by SI and iron-dependent ferroptosis was identified as the main cell death mechanism. Ferroptotic cell death depends on the Fenton reaction where H
O
and iron react to generate hydroxyl radicals which trigger lipid peroxidation. Our findings clearly provide evidence for superoxide dismutase (SOD) driven H
O
production fostering the Fenton reaction as indicated by triggered SOD activity upon DMOG + SI treatment as well as by reduced cell death levels upon SOD2 knockdown. In addition, iron transporters involved in non-transferrin-bound Fe
import as well as intracellular iron levels were also upregulated. Consequently, chelation of Fe
by 2'2-Bipyridyl completely rescued cells. Taken together, we show for the first time that HIF stabilization under oxidative stress conditions aggravates ferroptotic cell death in RPE cells. Thus, our study provides a novel link between hypoxia, oxidative stress and iron metabolism in AMD pathophysiology. Since iron accumulation and altered iron metabolism are characteristic features of AMD retinas and RPE cells, our cell culture model is suitable for high-throughput screening of new treatment approaches against AMD.
Life underground has shaped the auditory sense of subterranean mammals, shifting their hearing range to low frequencies. Mole-rats of the genus
have, however, been suggested to hear at frequencies up ...to 18.5 kHz, unusually high for a subterranean rodent. We present audiograms of three mole-rat species,
,
and the giant mole-rat
, based on evoked auditory brainstem potentials. All species showed low sensitivity and restricted hearing ranges at 60 dB SPL extending from 125 Hz to 4 kHz (5 octaves) with most-sensitive hearing between 0.8 kHz and 1.4 kHz. The high-frequency cut-offs are the lowest found in mammals to date. In contrast to predictions from middle ear morphology,
did not show higher sensitivity than
in the low-frequency range. These data suggest that the hearing range of
mole-rats is highly restricted to low frequencies and similar to that of other subterranean mammals.
Sexual activity and/or reproduction are associated with a doubling of life expectancy in the long-lived rodent genus
. To investigate the molecular mechanisms underlying this phenomenon, we analyzed ...636 RNA-seq samples across 15 tissues. This analysis suggests that changes in the regulation of the hypothalamic-pituitary-adrenal stress axis play a key role regarding the extended life expectancy of reproductive vs. non-reproductive mole-rats. This is substantiated by a corpus of independent evidence. In accordance with previous studies, the up-regulation of the proteasome and so-called 'anti-aging molecules', for example, dehydroepiandrosterone, is linked with enhanced lifespan. On the other hand, several of our results are not consistent with knowledge about aging of short-lived model organisms. For example, we found the up-regulation of the insulin-like growth factor 1/growth hormone axis and several other anabolic processes to be compatible with a considerable lifespan prolongation. These contradictions question the extent to which findings from short-lived species can be transferred to longer-lived ones.
ABSTRACT
Naked mole‐rats express many unusual traits for such a small rodent. Their morphology, social behaviour, physiology, and ageing have been well studied over the past half‐century. Many early ...findings and speculations about this subterranean species persist in the literature, although some have been repeatedly questioned or refuted. While the popularity of this species as a natural‐history curiosity, and oversimplified story‐telling in science journalism, might have fuelled the perpetuation of such misconceptions, an accurate understanding of their biology is especially important for this new biomedical model organism. We review 28 of these persistent myths about naked mole‐rat sensory abilities, ecophysiology, social behaviour, development and ageing, and where possible we explain how these misunderstandings came about.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
African mole-rats are subterranean rodents inhabiting underground burrows. This habitat entails risks of overheating, hypoxia, and scarce food availability. Consequently, many subterranean species ...have evolved low basal metabolism and low body temperature, but the regulation of these traits at the molecular level were unknown. Measurements of serum thyroid hormone (TH) concentrations in African mole-rats have revealed a unique TH phenotype, which deviates from the typical mammalian pattern. Since THs are major regulators of metabolic rate and body temperature, we further characterised the TH system of two African mole-rat species, the naked mole-rat (Heterocephalus glaber) and the Ansell's mole-rat (Fukomys anselli) at the molecular level in a comparative approach involving the house mouse (Mus musculus) as a well-studied laboratory model in TH research. Most intriguingly, both mole-rat species had low iodide levels in the thyroid and naked mole-rats showed signs of thyroid gland hyperplasia. However, contrary to expectations, we found several species-specific differences in the TH systems of both mole-rat species, although ultimately resulting in similar serum TH concentrations. These findings indicate a possible convergent adaptation. Thus, our study adds to our knowledge for understanding adaptations to the subterranean habitat.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The thyroid hormones (TH) triiodothyronine (T3) and its prohormone thyroxine (T4) are crucial for retinal development and function, and increasing evidence points at TH dysregulation as a cause for ...retinal degenerative diseases. Thus, precise regulation of retinal TH supply is required for proper retinal function, but knowledge on these mechanisms is still fragmentary. Several transmembrane transporters have been described as key regulators of TH availability in target tissues of which the monocarboxylate transporter 8 (MCT8), a high affinity transporter for T4 and T3, plays an essential role in the central nervous system. Moreover, in the embryonic chicken retina, MCT8 is highly expressed, but the postnatal availability of MCT8 in the mammalian retina was not reported to date. In the present study, spatiotemporal retinal MCT8 availability was examined in mice of different age. For this purpose, we quantified expression levels of Mct8 via Real-Time Reverse-Transcriptase PCR in mouse eyecups (C57BL/6) of juvenile and adult age groups. Additionally, age-dependent MCT8 protein levels were quantified via Western blotting and localized via immunofluorescence confocal microscopy. While no difference in Mct8 expression levels could be detected between age groups, MCT8 protein levels in juvenile animals were about two times higher than in adult animals based on Western blot analyses. Immunohistochemical analyses showed that MCT8 immunoreactivity in the eyecup was restricted to the retina and the retinal pigment epithelium. In juvenile mice, MCT8 was broadly observed along the apical membrane of the retinal pigment epithelium, tightly surrounding photoreceptor outer segments. Distinct immunopositive staining was also detected in the inner nuclear layer and the ganglion cell layer. However, in adult specimens, immunoreactivity visibly declined in all layers, which was in line with Western blot analyses. Since MCT8 was abundantly present in juvenile and about twofold lower in adult retinae, our findings suggest a pivotal role of MCT8 especially during postnatal maturation. The present study provides novel insights into age-dependent retinal TH supply, which might help to understand different aspects regarding retinal development, function, and disorders.
About 10% of all rodent species have evolved a subterranean way of life, although life in subterranean burrows is associated with harsh environmental conditions that would be lethal to most animals ...living above ground. Two key adaptations for survival in subterranean habitats are low resting metabolic rate (RMR) and core body temperature (T
). However, the upstream regulation of these traits was unknown thus far. Previously, we have reported exceptionally low concentrations of the thyroid hormone (TH) thyroxine (T4), and peculiarities in TH regulating mechanisms in two African mole-rat species, the naked mole-rat and the Ansell's mole-rat.
In the present study, we treated Ansell's mole-rats with T4 for four weeks and analyzed treatment effects on the tissue and whole organism level with focus on metabolism and thermoregulation.
We found RMR to be upregulated by T4 treatment but not to the extent that was expected based on serum T4 concentrations. Our data point towards an extraordinary capability of Ansell's mole-rats to effectively downregulate TH signaling at tissue level despite very high serum TH concentrations, which most likely explains the observed effects on RMR. On the other hand, body weight was decreased in T4-treated animals and T
was upregulated by T4 treatment. Moreover, we found indications of the hypothalamus-pituitary-adrenal axis potentially influencing the treatment effects.
Taken together, we provide the first experimental evidence that the low serum T4 concentrations of Ansell's mole-rats serve as an upstream regulator of low RMR and Tb. Thus, our study contributes to a better understanding of the ecophysiological evolution of the subterranean lifestyle in African mole-rats.
Many aging-associated physiological changes are known to occur in short- and long-lived species with different trajectories. Emerging evidence suggests that numerous life history trait differences ...between species are based on interspecies variations in gene expression. Little information is available, however, about differences in transcriptome changes during aging between mammals with diverging lifespans. For this reason, we studied the transcriptomes of five tissue types and two age cohorts of two similarly sized rodent species with very different lifespans: laboratory rats (
) and giant mole-rats (
), with maximum lifespans of 3.8 and more than 20 years, respectively. Our findings show that giant mole-rats exhibit higher gene expression stability during aging than rats. Although well-known aging signatures were detected in all tissue types of rats, they were found in only one tissue type of giant mole-rats. Furthermore, many differentially expressed genes that were found in both species were regulated in opposite directions during aging. This suggests that expression changes which cause aging in short-lived species are counteracted in long-lived species. Taken together, we conclude that expression stability in giant mole rats (and potentially in African mole-rats in general) may be one key factor for their long and healthy life.
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