Automated longitudinal monitoring of in vivo protein aggregation in neurodegenerative disease C. elegans models

Background: While many biological studies can be performed on cell-based systems, the investigation of molecular pathways related to complex human dysfunctions - e.g. neurodegenerative diseases - often requires long-term studies in animal models. The nematode Caenorhabditis elegans represents one of the best model organisms for many of these tests and, therefore, versatile and automated systems for accurate time-resolved analyses on C. elegans are becoming highly desirable tools in the field. Results: We describe a new multi-functional platform for C. elegans analytical research, enabling automated worm isolation and culture, reversible worm immobilization and long-term high-resolution imaging, and this under active control of the main culture parameters, including temperature. We employ our platform for in vivo observation of biomolecules and automated analysis of protein aggregation in a C. elegans model for amyotrophic lateral sclerosis (ALS). Our device allows monitoring the growth rate and development of each worm, at single animal resolution, within a matrix of microfluidic chambers. We demonstrate the progression of individual protein aggregates, i.e. mutated human superoxide dismutase 1 - Yellow Fluorescent Protein (SOD1-YFP) fusion proteins in the body wall muscles, for each worm and over several days. Moreover, by combining reversible worm immobilization and on-chip high-resolution imaging, our method allows precisely localizing the expression of biomolecules within the worms' tissues, as well as monitoring the evolution of single aggregates over consecutive days at the sub-cellular level. We also show the suitability of our system for protein aggregation monitoring in a C. elegans Huntington disease (HD) model, and demonstrate the system's ability to study long-term doxycycline treatment-linked modification of protein aggregation profiles in the ALS model. Conclusion: Our microfluidic-based method allows analyzing in vivo the long-term dynamics of protein aggregation phenomena in C. elegans at unprecedented resolution. Pharmacological screenings on neurodegenerative disease C. elegans models may strongly benefit from this method in the near future, because of its full automation and high-throughput potential

An automated microfluidic platform for long-term high-resolution imaging of C. elegans

We describe a microfluidic platform for the automated culture, treatment and long-term highresolution imaging of Caenorhabditis elegans nematodes under normal physiological conditions. Our device features: (i) a microfluidic design tailored for the isolation of L4 larvae from a mixed larval population and for successive culture and treatment; (ii) a worm immobilization method, based on the thermo-reversible gelation of a biocompatible polymer inside the microfluidic chip, thereby enabling high-resolution imaging; (iii) an integrated temperature control system, both to ensure viable environmental conditions for C. elegans culture and to steer the worm immobilization/release process

Long-term culture and high-resolution imaging of C. elegans using an automated microfluidic platform

The nematode Caernohabditis elegans is one of the most employed small model organisms in biology. Studies on transgenic animals often require the accurate observation of highly localized fluorescent signals inside the worms at high magnification, hence demanding the full immobilization of the animals. Moreover, in order to observe the dynamics of biological processes over the lifespan of a worm, the same worm has to be immobilized repeatedly, in a reversible manner and under normal physiological conditions. We describe a microfluidic platform for the automated culture, treatment and long-term high-resolution imaging of C. elegans. Our device features: (i) a microfluidic design tailored for the isolation of L4 larvae from a mixed larval population and for their successive culture and treatment; (ii) a worm immobilization method, based on the thermoreversible sol-gel transition of the biocompatible triblock copolymer Pluronic F127 inside the microfluidic chip, thereby enabling high-resolution imaging; (iii) an integrated temperature control system, both to ensure viable environmental conditions for C. elegans culture and to steer the worm immobilization/release process. We apply this device to observe mitochondrial dynamics in muscle cells during aging at single worm resolution. We expect our platform to enable the simultaneous study and repeated observation of multiple phenotypes in single worms or specific worm populations, such as lifespan and motility assays, in addition to high-resolution imaging

Characterization of different Pluronic (PF127) solutions.

<p>(A) PF127 viscosity as a function of temperature for a range of concentrations, measured with a cone-plate viscometer. The viscosity curves show a sharp rise at a specific temperature, corresponding to the sol-gel transition. (B) The viscosity at 25°C follows a quadratic progression as a function of PF127 concentration. The sol-gel transition temperature decreases linearly with increasing Pluronic concentration.</p

<i>C</i>. <i>elegans</i> immobilization protocol using a hydrogel-microbead matrix.

<p>(A) Worms are transferred from an agar plate into a droplet of liquid S Medium on a glass slide. (B) Precooled (~4°C) liquid Pluronic-microbead suspension is applied on the glass slide around the S Medium (red trace) and on a coverslip (not shown here). (C) The coverslip is positioned upside down and worms are immobilized in the gel-microbead matrix (red) in between the two glass substrates after thermalization (<i>T</i> ≈ 25°C). (D) Schematics of the worm immobilization technique with microbead spacers.</p

Gel-based immobilization of other small organisms.

<p>(A) Image of an immobilized <i>D</i>. <i>melanogaster</i> 1^st instar larva (length ~2 mm). (B) Image of an immobilized <i>T</i>. <i>brucei</i> unicellular parasite (length ~20 μm). (C) Pluronic hydrogel concentration-related reduction of head thrashes for <i>D</i>. <i>melanogaster</i> 1^st instar larvae, <i>D</i>. <i>melanogaster</i> larvae in the prepupal stage (n = 15 for each group), and reduction of body thrashes (swimming frequency) for <i>T</i>. <i>brucei</i> (n = 20). Data is present as mean±SD, * p≤0.05, ** p≤0.01, *** p≤0.001 and **** p≤0.0001 <i>vs</i> each 0% w/v Pluronic group, n = 15–20 for each group.</p

Evaluation of <i>C</i>. <i>elegans</i> immobilization and recovery for different conditions.

<p>(A-F) Pairs of successive bright field images (10× objective) give an indication of adult worm motility under different conditions: (A-C) in buffer solution (one stroke intervals) and (D-F) in a PF127 (30% w/v) hydrogel matrix (using false colors to visualize two successive snapshots (5 s intervals). 3 different degrees of worm compression have been applied in either case: (A, D) no compression (no microbeads), and vertical confinement defined by 40 μm (B, E) or 30 μm (C, F) spacer microbeads, respectively. Rectangles indicate regions of interest for high-resolution imaging. Displacement amplitudes of 3 distinct worm body regions (<i>s</i>_<i>bc</i> head close to the buccal cavity, <i>s</i>_<i>m</i> mid-region and <i>s</i>_<i>t</i> tail), as indicated in (A) or (D), were measured. (G) Mean amplitude of displacement over 5 s intervals of the 3 worm body regions, in buffer solution or in PF127 under different conditions: no beads/no compression (nb), and with 40 μm or 30 μm spacer beads. Data is presented as mean±SD, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001 <i>vs</i>. nb in each condition, n = 15 for each group. (H) Fluorescent image of mitochondria aggregates in a <i>Pmyo-3</i>::<i>mito</i>::<i>GFP</i> worm immobilized in a PF127-microbead (30 μm) matrix. A selected mitochondrial structure in mid-body region close to vulva is indicated by crosshairs (63× objective). (I) Mean absolute displacement over 5 s intervals of different selected mitochondrial structures near terminal bulb (<i>s</i>_<i>tb</i>), vulva in the mid-body region (<i>s</i>_<i>m</i>) and anus (<i>s</i>_<i>t</i>). Animals were immobilized in a PF127-microbead (30 μm) matrix. Data is presented as mean±SD, n = 10 for each group. (J) Frequency of repeating motion patterns for freely moving adult worms (thrashes in buffer) and during immobilization under different conditions in PF127 (mainly forward-backward motion). Data is presented as mean±SD, **** p≤ 0.0001 <i>vs</i> buffer group, n = 15 for each group. (K) Evaluation of worm recovery from short-term (10 min, orange bars) and long-term (1 hour, blue bars) immobilization in PF127 (30% w/v) and tetramisole (1mM). The thrashing frequency was measured 10 min, 3 h and 1 day after release, respectively. The control population was maintained in buffer solution (no immobilization) all the time (ctl, grey bar). Data is presented as mean±SD; for short-term immobilization, ***p≤ 0.001 <i>vs</i> ctl group; for long-term immobilization, *p≤ 0.05 and ****p≤ 0.0001 <i>vs</i> ctl group. N = 15 for each group. (L) Evaluation of worm fertility (N2 wild type) after long-term immobilization (1 hour and 2 hours) in PF127 (30% w/v) and using tetramisole (1mM, 1 hour) at 20°C. Measurements were made at adult stage at day 1, 2 or 3 after release. Data is presented as mean±SD, *p≤ 0.05 and **p≤ 0.01 <i>vs</i> ctl group of each day, n = 10 for each group.</p