Fig 2: Study design and methods. (A) Woodchucks were infected with woodchuck hepatitis virus (WHV) as newborns. At the age of 18–24 months, woodchucks were imaged with computed tomography. Ultrasound guided cryoablation was performed on three animals. Cryoablated woodchucks were imaged and euthanized 14 days after treatment. Whole blood was collected, liver and tumors were sectioned and formalin preserved, and peripheral blood mononuclear cells (PBMC) were purified. (B) Tumors were stained for CD3, CD4, NCAM and FOXP3 markers. Immunohistochemical quantification was performed on 5 distinct regions. Three regions were in the ablated tumors: 1. Ablation margin 2. Ablation zone, and 3. Unablated region. The fourth region was located within a separate, untreated tumor (UT) in ablated animals. The fifth region was located in tumors within control, untreated animals (CT) n=9. (C) PBMC were purified from whole blood, labeled, cultured with immune-modulating drugs for 4 days and stained with CD3 and CD4 antibodies. FACS analysis was performed and data analyzed for cell replication (n=3 cryoablated animals, n=5 control infected animals, n=2 control uninfected animals).

Fig 3: MitoSNO attenuates denervation of NMJTo elucidate the consequence of MitoSNO treatment on NMJ innervation and muscle injury we subjected ischemic hindlimb muscle to saline or MitoSNO treatment prior to release tourniquet for reperfusion. with sham treated mice as control. (a) Representative confocal images and quantification of percentage of denervated NMJs 3 h and 14 days following IR (scale bar = 20 μm, *** denotes p < 0.001, n = 5–6); (b) Representative confocal images of transverse sections of skeletal muscle expressing Ncam (red), Laminin (green), and DAPI (blue) and quantification of the percentage Ncam positive fibers at 14 days following injury (scale bar = 100 μm, * denotes p < 0.05, n = 4); (c) Serum creatine kinase activity 3 h following IR (*** denotes p < 0.001, n = 6); (d) Representative images of H&E stained transverse sections of skeletal muscle and quantification of percentage of fibers with centralized nuclei 14 days following IR (scale bar = 50 μm, ** denotes p < 0.01, n = 4). Data are represented as mean ± SEM.

Fig 4: Local immune effects evaluated with immune cell distribution and quantification within the cryolesions. Rows demonstrate immunohistochemical expression and quantification of immune infiltrates, from top to bottom: CD3+, CD4+, NCAM (natural killers’ cells) and FOXP3 (T-regulatory cells). Whole mount sections for each stain are shown in the left column with magnification of the cryolesion margin zone in the center column. Quantification for each immune stain by regions as defined in methods and Figure 1B is represented on the right column. Control animals N=5, cryoablated animals N=3, 1–2 sections per animal, p-values for the entire test are shown on the graphs. P-values between pairs of regions are summarized in Table 1.

Fig 5: Identification and purification of five human fetal musculoskeletal lineages. a Strategy for assessing lineage diversification during human development. For all populations, cells were isolated as lineage (LIN) negative (CD235a−CD45−CD31−). b Chondrocytes were sorted as LIN−CD34−BMPR1B+ cells. BMPR1B positively labels the superficial zone of fetal articular cartilage. c Osteoblasts were sorted as LIN−ALP+ cells. ALP (alkaline phosphatase) positively labels endochondral osteoblasts in fetal trabecular bone. d Myoblasts were isolated based on LIN−CD56+CD146+ expression. Co-expression of CD56 (red) and CD146 (green) in fetal myoblasts was confirmed by immunofluorescence. e Ligamentocytes and f tenocytes were depleted of LIN+ cells following digestion of anterior/posterior cruciate ligament and Achilles tendons, respectively. N = 3–4; scale bars = 50 µm