Preparing ductal epithelial organoids for high-spatial-resolution molecular profiling using mass spectrometry imaging.

Organoid culture systems are self-renewing, three-dimensional (3D) models derived from pluripotent stem cells, adult derived stem cells or cancer cells that recapitulate key molecular and structural characteristics of their tissue of origin. They generally form into hollow structures with apical-basolateral polarization. Mass spectrometry imaging (MSI) is a powerful analytical method for detecting a wide variety of molecules in a single experiment while retaining their spatiotemporal distribution. Here we describe a protocol for preparing organoids for MSI that (1) preserves the 3D morphological structure of hollow organoids, (2) retains the spatiotemporal distribution of a vast array of molecules (3) and enables accurate molecular identification based on tandem mass spectrometry. The protocol specifically focuses on the collection and embedding of the organoids in gelatin, and gives recommendations for MSI-specific sample preparation, data acquisition and molecular identification by tandem mass spectrometry. This method is applicable to a wide range of organoids from different origins, and takes 1 d from organoid collection to MSI data acquisition.

Oxygen-Dependent Lipid Profiles of Three-Dimensional Cultured Human Chondrocytes Revealed by MALDI-MSI.

Articular cartilage is exposed to a gradient of oxygen levels ranging from 5% at the surface to 1% in the deepest layers. While most cartilage research is performed in supraphysiological oxygen levels (19-21%), culturing chondrocytes under hypoxic oxygen levels (≤8%) promotes the chondrogenic phenotype. Exposure of cells to various oxygen levels alters their lipid metabolism, but detailed studies examining how hypoxia affects lipid metabolism in chondrocytes are lacking. To better understand the chondrocyte's behavior in response to oxygen, we cultured 3D pellets of human primary chondrocytes in normoxia (20% oxygen) and hypoxia (2.5% oxygen) and employed matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) in order to characterize the lipid profiles and their spatial distribution. In this work we show that chondrocytes cultured in hypoxia and normoxia can be differentiated by their lipid profiles. Among other species, phosphatidylglycerol species were increased in normoxic pellets, whereas phosphatidylinositol species were the most prominent lipids in hypoxic pellets. Moreover, spatial mapping revealed that phospahtidylglyycerol species were less prominent in the center of pellets where the oxygen level is lower. Additional analysis revealed a higher abundance of the mitochondrial-specific lipids, cardiolipins, in normoxic conditions. In conclusion MALDI-MSI described specific lipid profiles that could be used as sensors of oxygen level changes and may especially be relevant for retaining the chondrogenic phenotype, which has important implications for the treatment of bone and cartilage diseases.

Pharmacological and methodological aspects of the separation-induced vocalization test in guinea pig pups; a systematic review and meta-analysis.

The separation-induced vocalization test in guinea pig pups is one of many that has been used to screen for anxiolytic-like properties of drugs. The test is based on the cross-species phenomenon that infants emit distress calls when placed in social isolation. Here we report a systematic review and meta-analysis of pharmacological intervention in the separation-induced vocalization test in guinea pig pups. Electronic databases were searched for original research articles, yielding 32 studies that met inclusion criteria. We extracted data on pharmacological intervention, animal and methodological characteristics, and study quality indicators. Meta-analysis showed that the different drug classes in clinical use for the treatment of anxiety disorders, have comparable effects on vocalization behaviour, irrespective of their mechanism of action. Of the experimental drugs, nociception (NOP) receptor agonists proved very effective in this test. Analysis further indicated that the commonly used read-outs total number and total duration of vocalizations are equally valid. With regard to methodological characteristics, repeated testing of pups as well as selecting pups with moderate or high levels of vocalization were associated with larger treatment effects. Finally, reporting of study methodology, randomization and blinding was poor and Egger's test for small study effects showed that publication bias likely occurred. This review illustrates the value of systematic reviews and meta-analyses in improving translational value and methodological aspects of animal models. It further shows the urgent need to implement existing publication guidelines to maximize the output and impact of experimental animal studies.

Systematic reviews of animal studies; missing link in translational research?

BACKGROUND: The methodological quality of animal studies is an important factor hampering the translation of results from animal studies to a clinical setting. Systematic reviews of animal studies may provide a suitable method to assess and thereby improve their methodological quality. OBJECTIVES: The aims of this study were: 1) to evaluate the risk of bias assessment in animal-based systematic reviews, and 2) to study the internal validity of the primary animal studies included in these systematic reviews. DATA SOURCES: We systematically searched Pubmed and Embase for SRs of preclinical animal studies published between 2005 and 2012. RESULTS: A total of 91 systematic reviews met our inclusion criteria. The risk of bias was assessed in 48 (52.7%) of these 91 systematic reviews. Thirty-three (36.3%) SRs provided sufficient information to evaluate the internal validity of the included studies. Of the evaluated primary studies, 24.6% was randomized, 14.6% reported blinding of the investigator/caretaker, 23.9% blinded the outcome assessment, and 23.1% reported drop-outs. CONCLUSIONS: To improve the translation of animal data to clinical practice, systematic reviews of animal studies are worthwhile, but the internal validity of primary animal studies needs to be improved. Furthermore, risk of bias should be assessed by systematic reviews of animal studies to provide insight into the reliability of the available evidence.

Protein tyrosine phosphatase receptor type R deficient mice exhibit increased exploration in a new environment and impaired novel object recognition memory.

Mouse gene Ptprr encodes multiple protein tyrosine phosphatase receptor type R (PTPRR) isoforms that negatively regulate mitogen-activated protein kinase (MAPK) signaling pathways. In the mouse brain, PTPRR proteins are expressed in cerebellum, olfactory bulb, hippocampus, amygdala and perirhinal cortex but their precise role in these regions remains to be determined. Here, we evaluated phenotypic consequences of loss of PTPRR activity and found that basal smell was normal for Ptprr(-/-) mice. Also, spatial learning and fear-associated contextual learning were unaffected. PTPRR deficiency, however, resulted in impaired novel object recognition and a striking increase in exploratory activity in a new environment. The data corroborate the importance of proper control of MAPK signaling in cerebral functions and put forward PTPRR as a novel target to modulate synaptic processes.

Reduced Euchromatin histone methyltransferase 1 causes developmental delay, hypotonia, and cranial abnormalities associated with increased bone gene expression in Kleefstra syndrome mice.

Haploinsufficiency of Euchromatin histone methyltransferase 1 (EHMT1), a chromatin modifying enzyme, is the cause of Kleefstra syndrome (KS). KS is an intellectual disability (ID) syndrome, with general developmental delay, hypotonia, and craniofacial dysmorphisms as additional core features. Recent studies have been focused on the role of EHMT1 in learning and memory, linked to the ID phenotype of KS patients. In this study we used the Ehmt1(+/-) mouse model, and investigated whether the core features of KS were mimicked in these mice. When comparing Ehmt1(+/-) mice to wildtype littermates we observed delayed postnatal growth, eye opening, ear opening, and upper incisor eruption, indicating a delayed postnatal development. Furthermore, tests for muscular strength and motor coordination showed features of hypotonia in young Ehmt1(+/-) mice. Lastly, we found that Ehmt1(+/-) mice showed brachycephalic crania, a shorter or bent nose, and hypertelorism, reminiscent of the craniofacial dysmorphisms seen in KS. In addition, gene expression analysis revealed a significant upregulation of the mRNA levels of Runx2 and several other bone tissue related genes in P28 Ehmt1(+/-) mice. Runx2 immunostaining also appeared to be increased. The mRNA upregulation was associated with decreased histone H3 lysine 9 dimethylation (H3K9me2) levels, the epigenetic mark deposited by Ehmt1, in the promoter region of these genes. Together, Ehmt1(+/-) mice indeed recapitulate KS core features and can be used as an animal model for Kleefstra syndrome. The increased expression of bone developmental genes in the Ehmt1(+/-) mice likely contributes to their cranial dysmorphisms and might be explained by diminished Ehmt1-induced H3K9 dimethylation.