Parabiosis in Mice: A Detailed Protocol

Wang

Journal of Autoimmunity

et al. 2016

There is a significant unmet need in the treatment of primary biliary cirrhosis (PBC) despite significant data on the effector pathways that lead to biliary duct damage. We focused attention on a murine model of PBC, the dominant negative transforming growth factor β receptor II (Tg) mice. To further define the pathways that lead to biliary pathology in these mice, we developed Tg mice deleted of CD4 cells (CD4−/−Tg). Interestingly, these mice developed more severe cholangitis than control Tg mice. These mice, which lack CD4 cells, manifested increased levels of IFN-γ produced by effector CD8 cells. It appears that increased cholangitis is due to the absence of CD4 Treg cells. Based on these data, we parabiosed CD4−/−Tg mice with established disease at 8–9 weeks of age with C57BL/6 control mice. Such parabiotic “twins” had a significant reduction in autoimmune cholangitis, even though they had established pathology at the time of surgery. We prepared mixed bone marrow chimera mice constructed from CD4−/−Tg and CD8−/− mice and not only was cholangitis improved, but a decrease in terminally differentiated CD8+ T effector cells in the presence of wild type CD4 cells was noted. In conclusion, “correcting” the CD4 T cell subset, even in the presence of pathogenic CD8 T cells, is effective in treating autoimmune cholangitis.

Section: Methodsmentioning

confidence: 99%

Successful treatment of murine autoimmune cholangitis by parabiosis: Implications for hematopoietic therapy

Wang

Journal of Autoimmunity

et al. 2016

“…9 Mice were anesthetized by isoflurane in O 2 . Toe pinch was used as an indicator of the pain response.…”

Section: Parabiosismentioning

confidence: 99%

Developmental Heterogeneity of Cardiac Fibroblasts Does Not Predict Pathological Proliferation and Activation

Ali

Ranjbarvaziri²,

Talkhabi³

et al. 2014

Circulation Research

Self Cite

273

312

Rationale: Fibrosis is mediated partly by extracellular matrix–depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown. Objective: We sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury. Methods and Results: We showed that Thy1 + CD45 − CD31 − CD11b − Ter119 − cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles. Conclusions: The cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response.

The Journal of Immunology

“…Parabiosis was developed as described previously (19,20). Briefly, the mice were anesthetized by i.p.…”

Section: Parabiosismentioning

confidence: 99%

“….+ An), where V is the hematoma volume, t is the slice thickness, and Ai is the area of bleeding. Hemoglobin content in the hematoma tissues was also measured to further quantify hematoma size (19). Blood (0, 2, 4, 8, 16, and 20 ml) was added to fresh brain homogenates, and the OD values of the samples were measured at 540 nm using a spectrophotometer (Thermo Multiskan, Pittsburgh, PA).…”

Section: Hematoma Measurementmentioning

confidence: 99%

A20 Ameliorates Intracerebral Hemorrhage–Induced Inflammatory Injury by Regulating TRAF6 Polyubiquitination

Meng

Zhao

Zhou

et al. 2017

Reducing excessive inflammation is beneficial for the recovery from intracerebral hemorrhage (ICH). Here, the roles and mechanisms of A20 (TNFAIP3), an important endogenous anti-inflammatory factor, are examined in ICH. A20 expression in the PBMCs of ICH patients and an ICH mouse model was detected, and the correlation between A20 expression and neurologic deficits was analyzed. A20 expression was increased in PBMCs and was negatively related to the modified Rankin Scale score. A20 expression was also increased in mouse perihematomal tissues. A20−/− and A20-overexpressing mice were generated to further analyze A20 function. Compared with wild-type (WT) mice, A20−/− and A20-overexpressing mice showed significant increases and decreases, respectively, in hematoma volume, neurologic deficit score, mortality, neuronal degeneration, and proinflammatory factors. Moreover, WT-A20−/− parabiosis was established to explore the role of A20 in peripheral blood in ICH injury. ICH-induced damage, including brain edema, neurologic deficit score, proinflammatory factors, and neuronal apoptosis, was reduced in A20−/− parabionts compared with A20−/− mice. Finally, the interactions between TRAF6 and Ubc13 and UbcH5c were increased in A20−/− mice compared with WT mice; the opposite occurred in A20-overexpressing mice. Enhanced IκBα degradation and NF-κB activation were observed in A20−/− mice, but the results were reversed in A20-overexpressing mice. These results suggested that A20 is involved in regulating ICH-induced inflammatory injury in both the central and peripheral system and that A20 reduces ICH-induced inflammation by regulating TRAF6 polyubiquitination. Targeting A20 may thus be a promising therapeutic strategy for ICH.