Research Article: An Integrated In Vitro Imaging Platform for Characterizing Filarial Parasite Behavior within a Multicellular Microenvironment

Date Published: November 20, 2014

Publisher: Public Library of Science

Author(s): Timothy Kassis, Henry M. Skelton, Iris M. Lu, Andrew R. Moorhead, J. Brandon Dixon, Edward Mitre.

Abstract: Lymphatic Filariasis, a Neglected Tropical Disease, is caused by thread-like parasitic worms, including B. malayi, which migrate to the human lymphatic system following transmission. The parasites reside in collecting lymphatic vessels and lymph nodes for years, often resulting in lymphedema, elephantiasis or hydrocele. The mechanisms driving worm migration and retention within the lymphatics are currently unknown. We have developed an integrated in vitro imaging platform capable of quantifying B. malayi migration and behavior in a multicellular microenvironment relevant to the initial site of worm injection by incorporating the worm in a Polydimethylsiloxane (PDMS) microchannel in the presence of human dermal lymphatic endothelial cells (LECs) and human dermal fibroblasts (HDFs). The platform utilizes a motorized controllable microscope with CO2 and temperature regulation to allow for worm tracking experiments with high resolution over large length and time scales. Using post-acquisition algorithms, we quantified four parameters: 1) speed, 2) thrashing intensity, 3) percentage of time spent in a given cell region and 4) persistence ratio. We demonstrated the utility of our system by quantifying these parameters for L3 B. malayi in the presence of LECs and HDFs. Speed and thrashing increased in the presence of both cell types and were altered within minutes upon exposure to the anthelmintic drug, tetramisole. The worms displayed no targeted migration towards either cell type for the time course of this study (3 hours). When cells were not present in the chamber, worm thrashing correlated directly with worm speed. However, this correlation was lost in the presence of cells. The described platform provides the ability to further study B. malayi migration and behavior.

Partial Text: Lymphatic Filariasis (LF) is the single largest world-wide source of secondary lymphedema [1] and is caused by adult parasitic nematodes that target and dwell in the lymphatic system. An estimated 120 million people in 73 countries are currently infected, and a further 1.4 billion live in areas where filariasis is endemic [2]. Of the 120 million people harboring the parasites, 90% have Wuchereria bancrofti, while Brugia malayi and Brugia timori infections account for the other 10% [3]. All three parasites use mosquitoes as transmission vectors [4]. Infection is initiated when the host-seeking mosquito deposits an infective third-stage larva (L3) on the skin of the host during the process of obtaining a blood meal. The infective larvae then penetrate the skin at the site of the bite, presumably guided by chemoattractants [5], and migrate to the lymphatic vessels and lymph nodes of the host where after 6–12 months they mature into adult worms. The adult worms may reside within the lymphatic system for years before the host shows any clinical manifestations such as lymphedema, hydrocele, elephantiasis, chyluria and compromised immunity [6]–[12]. Following mating in the lymphatics, the parasites release live progeny called microfilariae, which circulate in the bloodstream. These microfilariae can then be ingested by a mosquito during a blood meal, where they undergo development to form L2 and finally L3 larvae. Hence, the life cycle continues [7].

We demonstrated a platform for monitoring long-term nematode migration related behavior in a complex multicellular microenvironment that is potentially scalable for high through-put drug screening. The image acquisition system is flexible and surpasses most other published systems in acquisition capability [49] (See Table S1). The platform can be used with any nematode, including C. elegans, which are the most widely used model for studying nematode migration and behavior, since both tracking and analysis are independent of worm size and shape. Video is captured using a 640×480 pixel resolution camera but is capable of using any NI Vision compatible camera. Experiments were performed at a frame rate of 15 fps while the system is configurable to run at 60 fps without any reduction in resolution. The graphical user interface (GUI) is easy to use and requires minimal user intervention. The set-up is scalable to include any given number of lanes with the only limitation given by the minimum required dimensions of the lanes in order to encompass the given worm size and how much ‘blind time’ is acceptable between successive imaging cycles. From our experiments with the current device dimensions, the addition of each lane adds an average of 17 seconds of blind time as the algorithm has an additional lane to scan and image. While the software is only compatible with current Zeiss manufactured microscopes, due to the fact that we utilized the Zeiss microscope SDK, it does provide us with full control of every part of the microscope. Due to the modular design of the control VIs, we can easily add full control of the fluorescent filter wheel, objective, focus, illumination and dual-camera ports for experiments requiring more complex image acquisition workflows. The PDMS-based choice chamber provides a cheap and robust platform for nematode behavioral assays in which their interaction with various cellular environments would be of interest. Although the worms are capable of moving on the surface of the PDMS a three dimensional matrix environment would better recapitulate the migratory environment the worm must traverse to reach the lymphatic [50]–[52]. This setup would provide the benefit of creating a more defined concentration gradient of any potential chemo-attractants released by cells, however, it is uncertain whether L3 B. malayi have the capability of moving through such an environment.



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