Reproducibility and Characterization of Head Kinematics During a Large Animal Acceleration Model of Traumatic Brain Injury

Acceleration parameters have been utilized for the last six decades to investigate pathology in both human and animal models of traumatic brain injury (TBI), design safety equipment, and develop injury thresholds. Previous large animal models have quantified acceleration from impulsive loading forces (i.e., machine/object kinematics) rather than directly measuring head kinematics. No study has evaluated the reproducibility of head kinematics in large animal models. Nine (five males) sexually mature Yucatan swine were exposed to head rotation at a targeted peak angular velocity of 250 rad/s in the coronal plane. The results indicated that the measured peak angular velocity of the skull was 51% of the impulsive load, was experienced over 91% longer duration, and was multi- rather than uni-planar. These findings were replicated in a second experiment with a smaller cohort (N = 4). The reproducibility of skull kinematics data was mostly within acceptable ranges based on published industry standards, although the coefficients of variation (8.9% for peak angular velocity or 12.3% for duration) were higher than the impulsive loading parameters produced by the machine (1.1 vs. 2.5%, respectively). Immunohistochemical markers of diffuse axonal injury and blood–brain barrier breach were not associated with variation in either skull or machine kinematics, suggesting that the observed levels of variance in skull kinematics may not be biologically meaningful with the current sample sizes. The findings highlight the reproducibility of a large animal acceleration model of TBI and the importance of direct measurements of skull kinematics to determine the magnitude of angular velocity, refine injury criteria, and determine critical thresholds

Pregnancy and viral pathogenesis: exploring mechanisms of disease through mouse models

Pregnancy is a dynamic process associated with many different physiologic changes that are necessary to support the growth and development of the fetus. Multisystemic maternal adaptations, while essential for the establishment and maintenance of successful pregnancy, may also predispose pregnant women to heightened disease susceptibility and/or increased disease severity, in the context of certain infectious pathogens. Different pathogens are associated with altered risk of infection or increased severity of disease, and can lead to obstetric complications. An understanding of the mechanisms that underlie altered infectious disease pathogenesis during pregnancy is essential for the discovery and improvement of diagnostics, therapeutics and prevention strategies. Because mechanistic studies cannot be readily performed in pregnant women, reliable mouse models are essential for these types of investigations. The studies in this dissertation characterize novel mouse models of two viral infections that occur during pregnancy, Zika (ZIKV) and influenza A virus (IAV), that are of significant public health importance. Infection of pregnant mice each of these viral pathogens results in differential disease pathogenesis that can lead to similar pregnancy outcomes. Intrauterine infection of pregnant mice with ZIKV results in no clinical disease in the dam, but infects and crosses the placenta to cause spontaneous abortion and congenital disease, including neuroinflammation and cortical thinning of neonates. Further, our studies in mice demonstrated that infection during earlier during gestation results in more severe disease outcome compared with infection later during pregnancy. In contrast, infection of pregnant mice with influenza virus resulted in severe maternal disease, characterized by a significant reduction in weight gain and hormonal dysregulation, often leading to complete pregnancy loss. Through a series of mechanistic studies, we ruled out pregnancy-associated pulmonary functional changes and pregnancy-associated estrogens as mediators of severe maternal disease, which were instead shown to be protective during influenza infection. Pregnancy-associated changes in pulmonary function, including increased lung compliance and total lung capacity in mice correlated with preserved pulmonary diffusing capacity during peak influenza disease. Similarly, treatment of nonpregnant female mice with pregnancy concentrations of the placental estrogen, estriol, conferred significant clinical protection during IAV infection, which was shown to be mediated through reduced immune cell recruitment to the lungs. Collectively, these data support the value of pregnant and hormone-manipulated mouse models for the study of viral pathogenesis. In addition to aiding in the mechanistic understanding of disease, these tractable systems can serve as platforms for testing novel preventative and therapeutic strategies, which can translate into pregnant women

Animal Models of Enterovirus D68 Infection and Disease

Human enterovirus D68 (EV-D68) is a globally reemerging respiratory pathogen that is associated with the development of acute flaccid myelitis (AFM) in children. Currently, there are no approved vaccines or treatments for EV-D68 infection, and there is a paucity of data related to the virus and host-specific factors that predict disease severity and progression to the neurologic syndrome. EV-D68 infection of various animal models has served as an important platform for characterization and comparison of disease pathogenesis between historic and contemporary isolates. Still, there are significant gaps in our knowledge of EV-D68 pathogenesis that constrain the development and evaluation of targeted vaccines and antiviral therapies. Continued refinement and characterization of animal models that faithfully reproduce key elements of EV-D68 infection and disease is essential for ensuring public health preparedness for future EV-D68 outbreaks

Progesterone (P4) increases amphiregulin (AREG) expression and administration of recombinant AREG protects P4-depleted female mice against IAV infection.

<p>Adult female mice were ovariectomized, treated with placebo (-P4) or exogenous P4 (+P4), and inoculated with a sublethal dose of IAV or mock-infected. The expression of amphiregulin (<i>Areg</i>) mRNA (A) and protein concentrations (B) in the lungs were quantified at 3, 5, 7, 9 and 14 dpi (n = 8-10/treatment/dpi). Gene expression was normalized to <i>Gapdh</i> and mock-infected controls using the ΔΔCt method. Ovariectomized mice were treated with placebo (-P4), placebo and recombinant amphiregulin (-P4 +rAREG), or P4 (+P4) and inoculated with a sublethal dose of IAV. To confirm AREG replacement, pulmonary concentrations of AREG were measured at 14 dpi (C). Mice were monitored daily for changes in body temperature (D) and clinical disease (E) (n = 9-10/treatment). H&E stained lung sections collected at 14 dpi were scored for inflammation as a cumulative score of perivasculitis, vasculitis, bronchiolitis, alveolitis, edema, consolidation, and necrosis (F). Representative images of overall inflammation (2X magnification) and focused areas (10X magnification) with cellular infiltration and edema are shown (G) (n = 3-5/treatment, with 10 fields per animal). Pulmonary function tests were performed at 14 dpi and lung diffusing capacity (DF_CO; H), lung compliance (Crs; I), and resistance (Rrs; J) were measured (n = 8-10/treatment). The dotted lines represent the value (means ±SEM) for mock-infected mice and bars and circles represent means ±SEM for IAV-infected mice from 2 independent experiments, with significant differences represented by asterisks (*).</p

Progesterone (P4) reduces inflammation and improves pulmonary function during sublethal IAV infection.

<p>Adult female mice were ovariectomized, treated with placebo (-P4) or exogenous P4 (+P4), and inoculated with a sublethal dose of IAV or mock-infected. Females (n = 23-25/treatment) were monitored daily for changes in rectal body temperature (A) and clinical disease (B) for 21 dpi. Infectious virus titers (C) and cell necrosis (D) were measured 3, 5, 7, 9 and 14 dpi (n = 5–10 per dpi). Percentage of lesioned areas (E), alveolitis scores (F), edema scores (G), and cumulative inflammation scores (H) were quantified in H&E stained lung sections at 14 and 25 dpi. The numbers of proliferating of Ki67+ cells were analyzed at 14 and 25 dpi and quantified using ImageJ (I) (n = 3-5/treatment/dpi with 10 fields per animal). Pulmonary function tests, measuring lung diffusing capacity (DF_CO; J), lung tissue compliance (Crs; K), and resistance (Rrs; L), were performed at 14 and 25 dpi with the dotted line representing the average value (mean ±SEM) for mock-infected mice (n = 7-10/treatment/dpi). Data represent means ±SEM from 2–3 independent experiments and significant differences are represented by asterisks (*).</p

Progesterone (P4) protects adult female mice against lethal IAV infection.

<p>Adult female mice were ovariectomized, treated with placebo (-P4) or exogenous P4 (+P4), and inoculated with lethal IAV or mock-infected. Serum was collected at 3, 5, 7, and 21 days post-inoculation (dpi) and P4 concentrations (mock n = 5, IAV n = 20–22 [i.e., n = 5–7 per dpi]) were analyzed by radioimmunoassay, and uterine horns (mock n = 13, IAV = 35–38 i.e., n = 12–14 dpi time-point) were weighed (A). Lungs were harvested at days 3, 5, or 7 dpi and mRNA expression of the progesterone receptor (<i>Pr</i>) was measured and normalized to GAPDH and mock-infected animals using the ΔΔCt method (B). Values for each measure (A and B) did not differ between dpi and are shown as aggregates. Mice (-P4 n = 20, +P4 n = 10) were monitored daily for changes in rectal body temperature (C) and survival (D) for 21 dpi. Infectious virus titers in the lungs were measured at 3, 5, or 7 dpi (E; n = 8-10/treatment/dpi). The correlation between changes in body temperature and virus titers at 7dpi, as a measure of disease tolerance, was quantified using a linear regression model (F; n = 12/treatment). H&E stained lung sections collected at 7dpi from mock-infected (G, panel 1 and 2) and IAV-infected females (G, panels 3–6) were scored for inflammation. Alveolitis (G panel 3 and 4, indicated by black triangles) and edema are shown (G panels 5 and 6, indicated by black stars), as well as corresponding histopathogical scores on a scale from 0–3 (H, n = 3/treatment, 10 fields/animal, 10X magnification). Data represent means ± SEM from two independent experiments and significant differences are represented by asterisks (*).</p

Progesterone-Based Therapy Protects Against Influenza by Promoting Lung Repair and Recovery in Females

<div><p>Over 100 million women use progesterone therapies worldwide. Despite having immunomodulatory and repair properties, their effects on the outcome of viral diseases outside of the reproductive tract have not been evaluated. Administration of exogenous progesterone (at concentrations that mimic the luteal phase) to progesterone-depleted adult female mice conferred protection from both lethal and sublethal influenza A virus (IAV) infection. Progesterone treatment altered the inflammatory environment of the lungs, but had no effects on viral load. Progesterone treatment promoted faster recovery by increasing TGF-β, IL-6, IL-22, numbers of regulatory Th17 cells expressing CD39, and cellular proliferation, reducing protein leakage into the airway, improving pulmonary function, and upregulating the epidermal growth factor amphiregulin (AREG) in the lungs. Administration of rAREG to progesterone-depleted females promoted pulmonary repair and improved the outcome of IAV infection. Progesterone-treatment of AREG-deficient females could not restore protection, indicating that progesterone-mediated induction of AREG caused repair in the lungs and accelerated recovery from IAV infection. Repair and production of AREG by damaged respiratory epithelial cell cultures <i>in vitro</i> was increased by progesterone. Our results illustrate that progesterone is a critical host factor mediating production of AREG by epithelial cells and pulmonary tissue repair following infection, which has important implications for women’s health.</p></div