Lesanpezeshki, Leila et al. (2021) International Worm Meeting "Burrrowing chip: A microfluidic platform to visualize and quantitate the burrowing behavior of C. elegans"

Anupom, Taslim, Laranjeiro, Ricardo, Norouzi Darabad, Masoud, Lesanpezeshki, Leila, Driscoll, Monica, Blawzdziewicz, Jerzy, A. Vanapalli, Siva

[International Worm Meeting, 2021]

Caenorhabditis elegans has been widely used to study the genetics of behavior and neuromuscular function. Most of the behavioral studies in C. elegans are limited to two-dimensional (2D) assessment of animal locomotion on agar plates (crawling) or in liquid (swimming), and it was not until recently that C. elegans studies began to focus on 3D motion. 3D locomotion where animals burrow through a matrix is known to be physiologically distinct from 2D crawling and swimming (Bilbao et al. 2018) and 3D movement evokes different gene expression responses (Hewitt et al. 2020). We have previously deployed a Pluronic hydrogel environment to assess the burrowing performance of muscle mutants. The stiffness of the gel can be tuned to challenge the animals to burrow through a higher mechanical resistance environment to increase the assay sensitivity (Lesanpezeshki et al. 2019). However, due to the assay geometry being in a well-plate, the full burrowing trajectory was not observable, and the animals could be only visualized at the top surface, precluding evaluation of the burrowing behavior throughout the gel layers. To address the above issues, we have developed a microfluidic platform called a burrowing chip, that enables visualization and assessment of burrowing behavior in the same Pluronic hydrogel environment. The microfluidic format provides excellent control of the chemotactic gradient and animal/gel loading. The shallow depth of the channel enables use of consumer cameras for visualizing and recording burrowing behavior. We show that the device provides a 3D environment to assess the animal's burrowing performance and the animal distribution along the channel during chemotaxis. Next, we identify two burrowing classes of behaviorally slow and fast movement, based on the time it takes animals to reach the attractant. Slow burrowers weakly maintain their direction toward the attractant. We have discovered pause intervals as a novel phenotype for stimulated burrowing animals, as the burrowing animals exhibit long pauses. Finally, we show that the burrowing chip is capable of characterizing the muscle mutants based on their lower burrowing velocity and higher frequency of pauses compared to wild-type animals. We suggest that the burrowing chip can be used to provide insights into the genetic basis of burrowing behavior and to assess the neuromuscular health in C. elegans, paving the road to identify therapeutic targets for age-related diseases and decline.

Rahman, Mizanur et al. (2017) International Worm Meeting "Microfluidics-based evaluation of neuromuscular healthspan in C.elegans."

Blawzdziewicz, Jerzy, Van-Bussel, Frank, Vanapalli, Siva, Szewczyk, Nathaniel, Edwards, Hunter, Rahman, Mizanur, Driscoll, Monica

[International Worm Meeting, 2017]

Aging studies in C.elegans based on functional metrics and molecular markers are essential to identify genetic and environmental determinants of healthspan. Although functional measures based on locomotion, pharyngeal pumping and oxidative stress have been used to interrogate healthspan of C.elegans, our understanding on maintenance and deterioration of the neuromuscular system with age is far from complete. Here, we propose muscle strength and stimulated reversals as two novel functional measures to report on the neuromuscular health of C.elegans. These measures are possible due to two technological advances made in our laboratory: NemaFlex - an image-based force-sensing micropillars that can quantify muscle strength and NemaLife - a microfluidics-based culturing apparatus that enables longitudinal aging experiments without the need for drug-induced blocking of progeny. We profiled strength across the lifespan of a wild-type population, and our results show that strength increases 5-fold from young adult to mid-life followed by a sharp decline - providing the first direct evidence of muscle strength loss due to age in C. elegans. With respect to stimulated reversals, we find that worms maintain a high reversal speed during their reproductive period and then declines at a rate that is nearly identical to the animal mortality rate. Interestingly, the decline in reversal speed precedes the onset of strength decline by about 3 days indicating that reversal speed is an early predictor of mortality. Moreover, since reversal speed is an indicator of motor activity, the early decline in reversal speed suggests that the presynaptic loss of function occurs early in life, consistent with prior electrophysiological studies. The stochastic nature of aging produces a wide distribution of lifespans in isogenic C. elegans populations. We observed that long lived worms generally maintain muscle strength long after median lifespan although their motor activity significantly declines. We observed four major classes of strength profiles among individual worms, each of which has a unique healthspan and motor activity pattern. For example, individuals starting with high reversal speed do not necessarily have long lifespan but all strong and long lived worms maintain very high motor activity during the late reproductive period. In summary, maintenance of strength together with motor activity is a possible route for increasing locomotory healthspan.

Rahman, Mizanur et al. (2015) International Worm Meeting "NemaFlex: A microfluidic tool for phenotyping (neuro)muscular strength in C. elegans."

Driscoll, Monica, Blawzdziewicz, Jerzy, Szewczyk, Nathaniel, Vanapalli, Siva A., Hewitt, Jennifer E., Van Bussel, Frank, Rahman, Mizanur

[International Worm Meeting, 2015]

Maintenance of muscle strength is essential for an individual's health and well-being. In fact, loss of muscle strength is a prognostic indicator for a variety of disorders including sarcopenia, cancer, and neuromuscular diseases. Decline in muscle strength is also a significant issue for space explorers. Therefore, a major challenge for muscle health research is to understand the genetic mechanisms regulating strength. C. elegans is an established genetic model that has muscle with genetic and physiological similarities to human muscle. Aging C. elegans also show muscle deterioration with age much like humans. Thus, prospective life-long muscle health studies can be accomplished in the roundworm C. elegans.To make C. elegans a comprehensive genetic model for muscle health investigations, we engineered NemaFlex-a microfluidic device containing deformable pillars for quantifying muscle strength in C. elegans. Animals crawl through the pillar array, causing pillar displacements from which local force data can be extracted. Since the forces fluctuate depending on the animal's behavior (velocity, body conformation, and position with respect to pillar), reproducible quantitation of animal strength has thus far remained elusive. We establish NemaFlex for high throughput muscle strength assays by developing a robust experimental protocol and analysis workflow to sample the full range of forces and define a metric for maximum strength. We also configure NemaFlex for recording strength across virtually the entire lifespan of animals permitting sarcopenia investigations.We find that forced contractions, such as induced by an acetylcholine agonist, show the same maximum strength as the untreated animals suggesting NemaFlex quantitates maximum strength. We tested mutants with neuronal defects (unc-17 and unc-119) and impaired sarcomeres (unc-52 and unc-112) and observe changes that verify the neuromuscular origin of strength. We profile strength across the lifespan of a wild-type population, and our results show that strength increases 7-fold from the young adult followed by a sharp late-age decline-providing the first direct evidence of muscle strength loss due to aging, similar to sarcopenia in humans. In summary, NemaFlex is a powerful tool to conduct prospective life-long investigations of muscle strength in C. elegans and mutants.

Rahman, Mizanur et al. (2015) International Worm Meeting "A microfluidic platform for parallelized aging and healthspan investigations in C. elegans."

Gabrilska, Rebecca, Rumbaugh, Kendra P., Edwards, Hunter, Szewczyk, Nathaniel, Driscoll, Monica, Birze, Nikolajs, Vanapalli, Siva A., Rahman, Mizanur, Hewitt, Jennifer E., Blawzdziewicz, Jerzy

[International Worm Meeting, 2015]

Caenorhabditis elegans has emerged as a model organism for aging research. Large-scale screens for longevity genes in C. elegans use liquid culture combined with drug-induced blocking of progeny, introducing physiological stress on the animals. Small-scale pilot screens adopt agar-based methods, which necessitates the tedious task of repetitively picking and transferring animals. In both approaches, it is practically impossible to add reagents or remove reagents (e.g., food or drugs) at multiple time points during the lifespan precluding detailed aging investigations. Finally, most screens do not score animals for multidimensional readouts of healthspan.We report a simple and high-throughput microfluidic platform addressing the limitations of current methods. The platform is capable of measuring lifespan and healthspan of wild type and mutants in parallel without a requirement for drug blocking of progeny production. Numerous trials have shown that we can remove progeny efficiently while retaining the adult animals in the device. To reduce physiological stress on the animals, we have optimized device geometry and feeding protocols such that the gait, body size, and lifespan are consistent with aging assays on agar. In addition to standard healthspan readouts such as locomotory prowess and pharyngeal pumping, the device allows recording of novel measures such as animal muscle strength and agility.We test the multifunctional capabilities of the device by conducting a pilot screen that includes wild-type, daf-2, daf-16, age-1 and eat-2 animals. We find that the lifespan curves of the wild-type and genetic mutants are consistent with the literature reports. We also show that in a targeted RNAi screen, the lifespan curves obtained were consistent with those of the genetic mutants. Analysis of muscle strength, agility, and locomotory prowess as a function of lifespan in the long-lived mutants reveal new insights into sarcopenia. In summary, we anticipate that the simplicity of our method, combined with unprecedented capacity to temporally manipulate the environment of the animals and record multidimensional healthspan measures will enable highly parallelized cross-sectional and longitudinal aging experiments.