Study No. 1

Title: Imaging Lyme Borreliosis In An Animal Model     

Background and significance.

While much is known about the role of ticks in the transmission of pathogenic agents, the biology of Borrelis burgdorferi in various mammalian hosts, and the multisystem infectious disease caused by this spirochete, virtually no information is available on the dynamics of Borrelia inoculation into the skin, the subsequent spirochete invasion of internal organs and the pathogenesis of neuroborreliosis. We propose to establish an animal model to study these events microscopically, at high resolution and in real-time. Based on our extensive studies over many years with a similar approach in malaria, we believe that these proposed studies are feasible at this time.

Experimental Design

Ticks. An Ixodes scapularis colony will be established to analyze B. burgdorferi transmission into skin.Fluorescent spirochetes. To visualize interactions between the spirochetes and various tissues of the infected host, we will obtain green and red fluorescent B. burgdorferi from Drs. Erol Fikrig and Justin Radolf and from Dr. Wolfram Zuckert, respectively.

Small animal host. To determine the optimal host, laboratory mice (C3H/HeJ, C57BI/6, Swiss Webster, SCID, nude) as well as gerbils will be infected as previously described. We will also obtain white footed mice (Peromyscus leucopus) from the Peromyscus Genetic Stock Center at the University of South Carolina to evaluate suitability of this natural reservoir host of B. burgdorferi for the planned studies.

In vitro cultivation and infection. Because microscopic examination of B. burgdorferi in infected tissues (other than the skin) requires a high density of organisms, we will grow the spirochetes in Barbour Stoenner-Kelly (BSK II) medium and infect the animals with large numbers of spirochetes. Various routes of infection (intracerebral, intradermal, intraperitoneal, and intravenous) will be compared to establish optimal experimental conditions for the efficient analysis.

Microscopic approaches. Digital wide field fluorescence microscopy, confocal microscopy, and two photon microscopy will be used for a comparative analysis of spirochete behavior in terms of resolution, depth of focus, and speed of data acquisition. The exact localization of spirochetes in the brain or other organs of interest, both in relation to tissue structure and infiltrating cells of the innate or adaptive immune system, will be determined by intravital imaging. Tissue sections will be used for histological staining and immunohistochemistry to monitor parameters such as development of microgliosis, infiltration of inflammatory and immune cells, secretion of cytokines, alterations of the blood rheology, etc. The use of mice with fluorescent endothelia, phagocytes, or dendritic cells will facilitate visualization of the tissue architecture and innate effector cells. Higher resolution can be obtained by electron microscopy; immunoelectron microscopy techniques will be used as appropriate.

Transmission into the skin. Dynamics of tick injection of spirochetes into the skin, the migration of these spirochetes through the skin, and the ways in which spirochetes leave skin to invade internal organs will be monitored in live mice. We will evaluate how immunization against Borrelia or against tick bites may affect spirochete transmission and their subsequent dynamics.

Neuroborreliosis and integrity of the blood-brain barrier(BBB). Brain will be analyzed for distribution of Borrelia. Markers for permeability of proteins (Evans blue or high molecular weight fluorescent dextran) or ions (Lissamine green B or sodium flluorescein) will be injected to determine damage to the BBB. Leakage of markers into the tissue will be quantified by spectrophotometry. To determine the viability of endothelial and other brain cells, we will inoculate combinations of nuclear dyes such as Hoechst, markers for apoptosis, and dead cell markers such as ethidium bromide. Changes in local reactive oxygen species (ROS) levels can be detected with reporter dyes such as DM-H2DCFDA.

Immunopathology. Similar to studies on cerebral malaria now being done in the lab, brains will be fixed and processed for analysis pf cerebral pathology, such as induction and release of cytokines and chemokines. Using commercial antibodies and molecular markers, we will determine the tissue distribution in response to infection of inducible nitric oxide synthase (iNOS) and pro-and anti-inflammatory cytokines such as TNF-alpha (including its receptor TNFR2), IL-1beta, IL-6, IL-8, LT-alpha, TGF-beta; adhesion molecules including ICAM-1; and various blood and immune cell populations including platelets, leukocytes, and CD8+ T cells.