Laser-Induced Porcine Model of Experimental Retinal Vein Occlusion:An Optimized Reproducible Approach

Retinal vein occlusion (RVO) is a frequent visually disabling condition. The management of RVO continues to challenge clinicians. Macular edema secondary to RVO is often recurrent, and patients typically require intravitreal injections for several years. Understanding molecular mechanisms in RVO is a key element in improving the treatment of the condition. Studying the molecular mechanisms in RVO at the retinal level is possible using animal models of experimental RVO. Most studies of experimental RVO have been sporadic, using only a few animals per experiment. Here, we report on 10 years of experience of the use of argon laser-induced experimental RVO in 108 porcine eyes from 65 animals, including 65 eyes with experimental branch retinal vein occlusion (BRVO) and 43 eyes with experimental central retinal vein occlusion (CRVO). Reproducibility and methods for evaluating and controlling ischemia in experimental RVO are reviewed. Methods for studying protein changes in RVO are discussed in detail, including proteomic analysis, Western blotting, and immunohistochemistry. Experimental RVO has brought significant insights into molecular changes in RVO. Testing intravitreal interventions in experimental RVO may be a significant step in developing personalized therapeutic approaches for patients with RVO

Proteome Analysis of Aflibercept Intervention in Experimental Central Retinal Vein Occlusion

Aflibercept is a frequently used inhibitor of vascular endothelial growth factor (VEGF) in the treatment of macular edema following central retinal vein occlusion (CRVO). Retinal proteome changes following aflibercept intervention in CRVO remain largely unstudied. Studying proteomic changes of aflibercept intervention may generate a better understanding of mechanisms of action and uncover aspects related to the safety profile. In 10 Danish Landrace pigs, CRVO was induced in both eyes with an argon laser. Right eyes were treated with intravitreal aflibercept while left control eyes received isotonic saline water. Retinal samples were collected 15 days after induced CRVO. Proteomic analysis by tandem mass tag-based mass spectrometry identified a total of 21 proteins that were changed in content following aflibercept intervention. In retinas treated with aflibercept, high levels of aflibercept components were reached, including the VEGF receptor-1 and VEGF receptor-2 domains. Fold changes in the additional proteins ranged between 0.70 and 1.19. Aflibercept intervention resulted in a downregulation of pigment epithelium-derived factor (PEDF) (fold change = 0.84) and endoplasmin (fold change = 0.91). The changes were slight and could thereby not be confirmed with less precise immunohistochemistry and Western blotting. Our data suggest that aflibercept had a narrow mechanism of action in the CRVO model. This may be an important observation in cases when macular edema secondary to CRVO is resistant to aflibercept intervention

Aflibercept Intervention in Experimental Branch Retinal Vein Occlusion Results in Upregulation of DnaJ Homolog Subfamily C Member 17

Aflibercept is an inhibitor of vascular endothelial growth factor (VEGF) used to treat macular edema following branch retinal vein occlusion (BRVO). Despite well-documented efficacy, there is limited knowledge about proteome changes following aflibercept intervention in BRVO. Proteome changes may provide insights into mechanisms of action as well as aspects related to safety profile. In seven Danish Landrace pigs, BRVO was induced with a well-established experimental model of argon laser-induced BRVO. BRVO was induced in both eyes. Three days after the induced BRVO, aflibercept was injected intravitreally in the right eyes, while the left eyes received intravitreal isotonic saline water. Retinas were collected 15 days after the induced BRVO and analyzed with label-free quantification liquid chromatography tandem mass spectrometry (LFQ LC-MS/MS). Fourteen proteins were changed in expression following aflibercept intervention in the BRVO model. LFQ LC-MS/MS identified an upregulation of DnaJ homolog subfamily C member 17 (DNAJC17) (fold change = 6.19) and a modest downregulation of isoform 2 of the protein encoded by N-myc downstream regulated gene 2 (NDRG2) (fold change = 0.40). NDRG2 was unchanged by Western blotting. In the additional significantly regulated proteins, only discrete changes were observed (fold changes 0.52–1.59). Our study is the first to report an association between aflibercept intervention and the heat shock protein DNAJC17. Our results indicate that the role of heat shock proteins in the treatment of BRVO should be further explored

Characterising Alzheimer's disease through integrative NMR- and LC-MS-based metabolomics

BackgroundAlzheimer's Disease (AD) is a complex and multifactorial disease and novel approaches are needed to illuminate the underlying pathology. Metabolites comprise the end-product of genes, transcripts, and protein regulations and might reflect disease pathogenesis. Blood is a common biofluid used in metabolomics; however, since extracellular vesicles (EVs) hold cell-specific biological material and can cross the blood-brain barrier, their utilization as biological material warrants further investigation. We aimed to investigate blood- and EV-derived metabolites to add insigts to the pathological mechanisms of AD. MethodsBlood samples were collected from 10 AD and 10 Mild Cognitive Impairment (MCI) patients, and 10 healthy controls. EVs were enriched from plasma using 100,000×g, 1 h, 4 °C with a wash. Metabolites from serum and EVs were measured using liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Multivariate and univariate analyses were employed to identify altered metabolites in cognitively impaired individuals. ResultsWhile no significant EV-derived metabolites were found differentiating patients from healthy individuals, six serum metabolites were found important; valine (p = 0.001, fold change, FC = 0.8), histidine (p = 0.001, FC = 0.9), allopurinol riboside (p = 0.002, FC = 0.2), inosine (p = 0.002, FC = 0.3), 4-pyridoxic acid (p = 0.006, FC = 1.6), and guanosine (p = 0.004, FC = 0.3). Pathway analysis revealed branched-chain amino acids, purine and histidine metabolisms to be downregulated, and vitamin B6 metabolism upregulated in patients compared to controls. ConclusionUsing a combination of LC-MS and NMR methodologies we identified several altered mechanisms possibly related to AD pathology. EVs require additional optimization prior to their possible utilization as a biological material for AD-related metabolomics studies

A 5700 year-old human genome and oral microbiome from chewed birch pitch

Abstract: The rise of ancient genomics has revolutionised our understanding of human prehistory but this work depends on the availability of suitable samples. Here we present a complete ancient human genome and oral microbiome sequenced from a 5700 year-old piece of chewed birch pitch from Denmark. We sequence the human genome to an average depth of 2.3× and find that the individual who chewed the pitch was female and that she was genetically more closely related to western hunter-gatherers from mainland Europe than hunter-gatherers from central Scandinavia. We also find that she likely had dark skin, dark brown hair and blue eyes. In addition, we identify DNA fragments from several bacterial and viral taxa, including Epstein-Barr virus, as well as animal and plant DNA, which may have derived from a recent meal. The results highlight the potential of chewed birch pitch as a source of ancient DNA

A 5700 year-old human genome and oral microbiome from chewed birch pitch

Abstract: The rise of ancient genomics has revolutionised our understanding of human prehistory but this work depends on the availability of suitable samples. Here we present a complete ancient human genome and oral microbiome sequenced from a 5700 year-old piece of chewed birch pitch from Denmark. We sequence the human genome to an average depth of 2.3× and find that the individual who chewed the pitch was female and that she was genetically more closely related to western hunter-gatherers from mainland Europe than hunter-gatherers from central Scandinavia. We also find that she likely had dark skin, dark brown hair and blue eyes. In addition, we identify DNA fragments from several bacterial and viral taxa, including Epstein-Barr virus, as well as animal and plant DNA, which may have derived from a recent meal. The results highlight the potential of chewed birch pitch as a source of ancient DNA