Automated monitoring of respiratory rate as a novel humane endpoint: A refinement in mouse metastatic lung cancer models.

In oncology research, while xenograft tumor models are easily visualized and humane endpoints can be clearly defined, metastatic tumor models are often based on more subjective clinical observations as endpoints. This study aimed at identifying objective non-invasive criteria for predicting imminent distress and mortality in metastatic lung tumor-bearing mice. BALB/c and C57BL/6 mice were inoculated with CT26 or B16F10 cells, respectively. The mice were housed in Vium smart cages to continuously monitor and stream respiratory rate and locomotion for up to 28 days until scheduled euthanasia or humane endpoint criteria were met. Body weight and body temperature were measured during the study. On days 11, 14, 17 and 28, lungs of subsets of animals were microCT imaged in vivo to assess lung metastasis progression and then euthanized for lung microscopic evaluations. Beginning at day 21, most tumor-bearing animals developed increased respiratory rates followed by decreased locomotion 1-2 days later, compared with the baseline values. Increases in respiratory rate did not correlate to surface tumor nodule counts or lung weight. Body weight measurement did not show significant changes from days 14-28 in either tumor-bearing or control animals. We propose that increases in respiratory rate (1.3-1.5 X) can be used to provide an objective benchmark to signal the need for increased clinical observations or euthanasia. Adoption of this novel humane endpoint criterion would allow investigators time to collect tissue samples prior to spontaneous morbidity or death and significantly reduce the distress of mice in the terminal stages of these metastatic lung tumor models.

Behavioral, Histopathologic, and Molecular Biological Responses of Nanoparticle- and Solution-Based Formulations of Vincristine in Mice.

Clinical use of the chemotherapeutic agent vincristine (VCR) is limited by chemotherapy-induced peripheral neuropathy (CiPN). A new formulation of VCR encapsulated by nanoparticles has been proposed and developed to alleviate CiPN. We hypothesized in nonclinical animals that the nanoparticle drug would be less neurotoxic due to different absorption and distribution properties to the peripheral nerve from the unencapsulated free drug. Here, we assessed whether VCR encapsulation in nanoparticles alleviates CiPN using behavioral gait analysis (CatWalk), histopathologic and molecular biological (RT-qPCR) approaches. Adult male C57BL/6 mice were assigned to 3 groups (empty nanoparticle, nano-VCR, solution-based VCR, each n = 8). After 15 days of dosing, animals were euthanized for tissue collection. It was shown that intraperitoneal administration of nano-VCR (0.15 mg/kg, every other day) and the empty nanoparticle resulted in no changes in gait parameters; whereas, injection of solution-based VCR resulted in decreased run speed and increased step cycle and stance (P < 0.05). There were no differences in incidence and severity of degeneration in the sciatic nerves between the nano-VCR-dosed and solution-based VCR-dosed animals. Likewise, decreased levels of a nervous tissue-enriched microRNA-183 in circulating blood did not show a significant difference between the nano- and solution-based VCR groups (P > 0.05). Empty nanoparticle administration did not cause any behavioral, microRNA, or structural changes. In conclusion, this study suggests that the nano-VCR formulation may alleviate behavioral changes in CiPN, but it does not improve the structural changes of CiPN in peripheral nerve. Nanoparticle properties may need to be optimized to improve biological observations.

Loss of Roundabout Guidance Receptor 2 (Robo2) in Podocytes Protects Adult Mice from Glomerular Injury by Maintaining Podocyte Foot Process Structure.

Roundabout guidance receptor 2 (ROBO2) plays an important role during early kidney development. ROBO2 is expressed in podocytes, inhibits nephrin-induced actin polymerization, down-regulates nonmuscle myosin IIA activity, and destabilizes kidney podocyte adhesion. However, the role of ROBO2 during kidney injury, particularly in mature podocytes, is not known. Herein, we report that loss of ROBO2 in podocytes [Robo2 conditional knockout (cKO) mouse] is protective from glomerular injuries. Ultrastructural analysis reveals that Robo2 cKO mice display less foot process effacement and better-preserved slit-diaphragm density compared with wild-type littermates injured by either protamine sulfate or nephrotoxic serum (NTS). The Robo2 cKO mice also develop less proteinuria after NTS injury. Further studies reveal that ROBO2 expression in podocytes is up-regulated after glomerular injury because its expression levels are higher in the glomeruli of NTS injured mice and passive Heymann membranous nephropathy rats. Moreover, the amount of ROBO2 in the glomeruli is also elevated in patients with membranous nephropathy. Finally, overexpression of ROBO2 in cultured mouse podocytes compromises cell adhesion. Taken together, these findings suggest that kidney injury increases glomerular ROBO2 expression that might compromise podocyte adhesion and, thus, loss of Robo2 in podocytes could protect from glomerular injury by enhancing podocyte adhesion that helps maintain foot process structure. Our findings also suggest that ROBO2 is a therapeutic target for podocyte injury and podocytopathy.

Nanoparticle contrast-enhanced micro-CT: A preclinical tool for the 3D imaging of liver and spleen in longitudinal mouse studies.

In drug discovery and development, X-ray micro-computed tomography (micro-CT) has gained increasing importance over the past decades. In recent years, micro-CT imaging of soft tissues has become popular due to the introduction of a variety of radiopaque contrast agents. More recently, nanoparticle-based ExiTron nano 12,000 has become commercially available for the nonclinical micro-CT imaging of soft tissues in rodents. Phagocytosis and accumulation of the contrast agent by Kupffer cells in the liver, as well as macrophages in the spleen, increase the soft tissue X-ray attenuation for up to 6 months. Therefore, it is essential to understand the potential toxicity of this nanomaterial in micro-CT imaging prior to its application in pharmacology and/or toxicology studies. Herein, we describe the time-course and distribution of the contrast in the liver, spleen and blood after a single intravenous injection (IV) of this nanoparticle contrast agent at 0.1 ml/mouse. Thoracic images of male adult C57BL/6 mice were acquired using a Bruker SkyScan 1276 micro-CT over a period of 29 days. The stability of X-ray attenuation enhancement in the above tissues was also tested after a single dose of Kupffer cell toxicant gadolinium chloride (GdCl3) at 15 mg/kg on day 2. The liver, spleen and kidney were examined microscopically on days 15 and 29 post treatment. Serum and liver cytokines (IL-1ß, IL-2, IL-6, IL-10, IL-12p70, IFN-γ, IP-10, MIP1-α, MIP1-ß and TNF-α) were quantified on days 15 and 29 as indicators of a pro-inflammatory response to treatment. This study determined that there was an accumulation of amphophilic granular material in the cells of the mononuclear phagocyte system in the liver and spleen following a single dose of ExiTron nano 12,000 and a second dose of GdCl3 or its vehicle. However, ExiTron nano12000 contrast administration did not cause any hepatotoxicity in the liver, nor did pro-inflammatory cytokines release in the liver or serum. Similarly, there were no adverse pathologies in the spleen or kidneys. In summary, ExiTron nano12000 contrast agent-enhanced micro-CT could be used as a safe method in up to 29-day longitudinal efficacy and toxicology mouse studies for the non-invasive assessment of the liver and spleen.