A Mouse Holder for Awake Functional Imaging in Unanesthetized Mice: Applications in 31P Spectroscopy, Manganese-Enhanced Magnetic Resonance Imaging Studies, and Resting-State Functional Magnetic Resonance Imaging.

Anesthesia is often used in preclinical imaging studies that incorporate mouse or rat models. However, multiple reports indicate that anesthesia has significant physiological impacts. Thus, there has been great interest in performing imaging studies in awake, unanesthetized animals to obtain accurate results without the confounding physiological effects of anesthesia. Here, we describe a newly designed mouse holder that is interfaceable with existing MRI systems and enables awake in vivo mouse imaging. This holder significantly reduces head movement of the awake animal compared to previously designed holders and allows for the acquisition of improved anatomical images. In addition to applications in anatomical T2-weighted magnetic resonance imaging (MRI), we also describe applications in acquiring 31P spectra, manganese-enhanced magnetic resonance imaging (MEMRI) transport rates and resting-state functional magnetic resonance imaging (rs-fMRI) in awake animals and describe a successful conditioning paradigm for awake imaging. These data demonstrate significant differences in 31P spectra, MEMRI transport rates, and rs-fMRI connectivity between anesthetized and awake animals, emphasizing the importance of performing functional studies in unanesthetized animals. Furthermore, these studies demonstrate that the mouse holder presented here is easy to construct and use, compatible with standard Bruker systems for mouse imaging, and provides rigorous results in awake mice.

MiR-590 Promotes Transdifferentiation of Porcine and Human Fibroblasts Toward a Cardiomyocyte-Like Fate by Directly Repressing Specificity Protein 1.

BACKGROUND: Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells represents a promising potential new therapy for treating heart disease, inducing significant improvements in postinfarct ventricular function in rodent models. Because reprogramming factors effective in transdifferentiating rodent cells are not sufficient to reprogram human cells, we sought to identify reprogramming factors potentially applicable to human studies. METHODS AND RESULTS: Lentivirus vectors expressing Gata4, Mef2c, and Tbx5 (GMT); Hand2 (H), Myocardin (My), or microRNA (miR)-590 were administered to rat, porcine, and human cardiac fibroblasts in vitro. induced cardiomyocyte-like cell production was then evaluated by assessing expression of the cardiomyocyte marker, cardiac troponin T (cTnT), whereas signaling pathway studies were performed to identify reprogramming factor targets. GMT administration induced cTnT expression in ≈6% of rat fibroblasts, but failed to induce cTnT expression in porcine or human cardiac fibroblasts. Addition of H/My and/or miR-590 to GMT administration resulted in cTNT expression in ≈5% of porcine and human fibroblasts and also upregulated the expression of the cardiac genes, MYH6 and TNNT2. When cocultured with murine cardiomyocytes, cTnT-expressing porcine cardiac fibroblasts exhibited spontaneous contractions. Administration of GMT plus either H/My or miR-590 alone also downregulated fibroblast genes COL1A1 and COL3A1. miR-590 was shown to directly suppress the zinc finger protein, specificity protein 1 (Sp1), which was able to substitute for miR-590 in inducing cellular reprogramming. CONCLUSIONS: These data support porcine studies as a surrogate for testing human cardiac reprogramming, and suggest that miR-590-mediated repression of Sp1 represents an alternative pathway for enhancing human cardiac cellular reprogramming.

Comparison of Cesium-137 and X-ray Irradiators by Using Bone Marrow Transplant Reconstitution in C57BL/6J Mice.

Mice are used extensively in transplantation studies involving bone marrow ablation. Due to the increasing security issues and expenses involved with γ irradiators, self-contained X-ray irradiators have been increasing in popularity. We hypothesized that bone marrow ablation by irradiation of mice with a (137)Cs irradiator would be comparable to that from an X-ray source irradiator. A lethal-dose curve was obtained by irradiating C57BL/6J mice with 500, 700, 900, and 1100 cGy from either source. These data were used to determine the lethal radiation exposure range for a noncompetitive bone marrow engraftment curve for each source. At 90 d after reconstitution, the bone marrow engraftment curves revealed significant differences between the 2 sources in the establishment of B cell, myeloid, and T cell lineages. Murine B cell reconstitution after exposure to a (137)Cs source was greater than that after X-ray exposure at each dose level, whereas the converse was true for myeloid cell reconstitution. At the 1050- and 1100-cGy doses, mice irradiated by using the X-ray source demonstrated higher levels of T cell reconstitution but decreased survival compared with mice irradiated with the (137)Cs source. We concluded that although both sources ablated endogenous bone marrow sufficiently to enable stem cell engraftment, there are distinct physiologic responses that should be considered when choosing the optimal source for use in a study and that irradiation from the (137)Cs source was associated with lower overall morbidity due to opportunistic infection.

Plasma and cerebrospinal fluid pharmacokinetics of thalidomide and lenalidomide in nonhuman primates.

PURPOSE: Thalidomide, originally developed as a sedative, was subsequently identified to have antiangiogenic properties. Lenalidomide is an antiangiogenic and immunomodulatory agent that has been utilized in the treatment of patients with brain tumors. We studied the pharmacokinetics and cerebrospinal fluid (CSF) penetration of thalidomide and lenalidomide in a nonhuman primate model. METHODS: A dose of 50 mg of thalidomide or 20 mg of lenalidomide was administered once orally to each of three rhesus monkeys. Plasma and CSF samples were obtained at specified intervals, and the thalidomide or lenalidomide concentrations were determined by high-performance liquid chromatography with tandem mass spectrometry. Pharmacokinetic parameters were estimated using noncompartmental methods. CSF penetration was calculated as area under the concentration-time curve (AUC) CSF/AUC plasma. RESULTS: For thalidomide, the median apparent clearance (Cl/F) was 2.9 mL/min/kg, the median plasma AUC was 80 µM h, and the median terminal half-life (t(½)) was 13.3 h. For lenalidomide, the median Cl/F was 8.7 mL/min/kg, the median AUC was 9 µM h, and the median t(½) was 5.6 h. Thalidomide was detected in the CSF of all animals, with a median penetration of 42%. Lenalidomide was detected in the CSF of 2 of 3 animals, with a CSF penetration of 11% in each. CONCLUSION: Thalidomide and lenalidomide penetrate into the CSF after oral administration of clinically relevant doses. Plasma exposure to lenalidomide was similar in our model to that observed in studies involving children who have brain tumors. These results support further development of lenalidomide for the treatment of central nervous system malignancies.

Plasma and cerebrospinal fluid pharmacokinetics of ABT-888 after oral administration in non-human primates.

PURPOSE: ABT-888 inhibits poly(ADP-ribose) polymerase (PARP) and may enhance the efficacy of chemotherapy and radiation in CNS tumors. We studied the plasma and cerebrospinal fluid (CSF) pharmacokinetics (PK) of ABT-888 in a non-human primate (NHP) model that is highly predictive of human CSF penetration. METHODS: ABT-888, 5 mg/kg, was administered orally to three NHPs. Serial blood and CSF samples were obtained. Plasma and CSF concentrations of ABT-888 were measured using LC/MS/MS, and the resulting concentration versus time data were evaluated using non-compartmental and compartmental PK methods. RESULTS: The CSF penetration of ABT-888 was 57+/-7% (mean+/-SD). The peak ABT-888 concentration in the plasma was 0.62+/-0.18 microM. Plasma and CSF AUC0-infinity were 3.7+/-1.7 and 2.1+/-0.8 microM h. PARP inhibition in peripheral blood mononuclear cells was evident 2 h after ABT-888 administration. CONCLUSION: The CSF penetration of ABT-888 after oral administration was 57%. Plasma and CSF concentrations were in the range that has been shown to inhibit PARP activity in vivo in humans.

Plasma and cerebrospinal fluid pharmacokinetics of tasidotin (ILX-651) and its metabolites in non-human primates.

PURPOSE: To evaluate the plasma and cerebrospinal fluid (CSF) pharmacokinetics and CSF penetration of tasidotin and metabolites in a nonhuman primate model. METHODS: Tasidotin 0.75 mg/kg was administered intravenously. The plasma and CSF concentrations of tasidotin and its metabolites were determined. Pharmacokinetic parameters were estimated using model-independent and model-dependent methods. RESULTS: The mean (+/-SD) CSF:plasma AUC ratio for tasidotin was 1.1 +/- 0.4. For tasidotin, tasidotin-C-carboxylate and desprolyl-tasidotin-C-carboxylate the plasma AUCs (mean +/- SD) were 30 +/- 10, 54 +/- 19 and 12 +/- 2 microM min, and apparent plasma half-lives were 27 +/- 4, 229 +/- 73 and 100 +/- 29 min. The plasma clearance of tasidotin was 44 +/- 14 ml/min/kg. The CSF AUC and half-life of tasidotin was 28 +/- 10 microM min and 96 +/- 40 min. The model-dependent plasma clearance was 35 ml/min/kg for tasidotin and 2 ml/min/kg for tasidotin-C-carboxylate. CONCLUSIONS: Tasidotin penetrates into the CSF well and further evaluation of its activity in the treatment of central nervous system malignancies should be considered.