Neurotoxicity evaluation of nanomaterials using C. elegans: survival, locomotion behaviors, and oxidative stress

Abstract

Nanomaterials are broadly used in variety of industries and consumer products. However, studies have demonstrated that many of these nanomaterials, including metal-containing nanoparticles and nanoplastics, have neurotoxic effects. Caenorhabditis elegans (C. elegans) is a widely used model organism with numerous advantages for research, including transparency, short life span, well characterized nervous system, complete connectome, available genome, and numerous genetic tools. C. elegans has been extensively used for neurotoxicity assessment of multiple chemicals via survival assays, behavioral tests, neuronal morphology studies, and various molecular and mechanistic analyses. Detailed protocols describing general assays in C. elegans to examine the neurotoxic effects of nanomaterials, however, are limited. Here, we describe protocols for the neurotoxicity assessment of nanomaterials in C. elegans. We describe the steps for both exposure, and subsequent evaluation of survival, locomotion behavior, and oxidative stress. Survival and locomotion behavior are measured in wildtype N2 strains to assess acute neurotoxicity. Oxidative stress is used as an endpoint here since it is one of the most predominant and common changes induced by nanomaterials. For oxidative stress assays, VP596 nematodes, which expresses GFP when skn-1 (the worm homolog of Nrf2) is activated, are evaluated in response to test nanomaterials. In all, these assays can be readily used to quickly examine the neurotoxicity of nanomaterials in vivo, laying the foundation for mechanistic studies of nanomaterials and their impacts on health and physiology.

Basic Protocol 1: Exposure of C. elegans to nanomaterials

Basic Protocol 2: Survival assessment

Basic Protocol 3: Assessment of locomotion behavior

Basic Protocol 4: Analysis of oxidative stress

INTRODUCTION:

Owing to their unique physical and chemical properties, nanomaterials have been used in many fields, including medical devices, therapeutics, imaging, food, and agriculture (Amiri & Shokrollahi, 2013; He, Deng, & Hwang, 2019; Oberdörster, Oberdörster, & Oberdörster, 2005). Despite their widespread use, adverse effects of nanomaterials have been reported, including neurotoxicity, immunotoxicity, reproductive and development toxicity, hepatotoxicity, and nephrotoxicity (Ema, Hougaard, Kishimoto, & Honda, 2016; Ju-Nam & Lead, 2008; Madannejad et al., 2019; Singh, 2019), highlighting the need for additional studies to understand how these materials can impact physiology.

Caenorhabditis elegans (C. elegans) is a model organism widely used in toxicity assessment due to many of its characteristics, such as transparency, short life span, and ease of culture (Hunt, 2017; Laura L. Maurer, Ian T. Ryde, Xinyu Yang, & Joel N. Meyer, 2015; Libânia Queirós et al., 2021). Furthermore, this organism exhibits a well conserved and fully characterized nervous system. Notably, it is the only model organism for which a complete connectome has recently been described (Cook et al., 2019), allowing to track every single neuron in a living animal. Moreover, the transparent nature of C. elegans provides researchers with the ability to visualize morphological changes in the nervous system via fluorescent proteins, making it well suited for observing changes due to toxicant insult in vivo (Ijomone, Miah, Akingbade, Bucinca, & Aschner, 2020). Therefore, C. elegans has been recognized as an ideal model for evaluating neurotoxicity induced by metals (Lawes et al., 2020), therapeutic chemicals (Wellenberg et al., 2021), engineered nanoparticles (Viau, Haçariz, Karimian, & Xia, 2020), pesticides (Lewis, Gehman, Baer, & Jackson, 2013; Salim & Rajini, 2014), and many other chemicals.

Commonly used laboratory tests for neurotoxicity evaluation in C. elegans include (a) lethality endpoints, i.e., survival; (b) behavioral endpoints, such as locomotion and foraging (P. Li, Xu, Wu, Lei, & He, 2017; Xu et al., 2017); (c) molecular endpoints, such as oxidative stress, acetylcholinesterase activity, and changes in gene expression (Mor et al., 2020; Zheng, Chen, Li, & Aschner, 2020), and (d) morphological and physiological endpoints, like neuronal structure and neurodegeneration (Latimer et al., 2022; L. Queirós et al., 2019). While many protocols are available to study these endpoints in various contexts, detailed protocols for the application of these methods to nanotoxicity assessment, particularly in neurotoxicology, remain scarce.

Here, we describe how to use C. elegans for simple and relatively quick neurotoxicity evaluation of nanomaterials of interest. Specifically, we describe assays to assess survival, oxidative stress, and locomotion in response to nanomaterials exposure. Survival is the most commonly used endpoint, as it is a direct indicator of a nanomaterials’ acute toxicity. It is also informative in dose selection for subsequent behavioral or mechanistic studies. Locomotion behavior is a classic index reflecting the functional state of motor neurons (Xu et al., 2017). Head thrash and body bend are the two mostly commonly used endpoints in locomotion evaluation and, thus, their assessment is described in this article. A defect in locomotion behavior can reflect dysregulation in dopaminergic (DAergic) signaling (Chen et al., 2015). Oxidative stress is one of the most common mechanisms proposed for nanomaterials-induced toxicity (Čapek & Roušar, 2021). Excess reactive oxygen species (ROS) can result in potential adverse effects, such as decline of survival, shortage of lifespan, and altered locomotion behavior (Horie & Tabei, 2021; Sinis, Gourgoulianis, Hatzoglou, & Zarogiannis, 2019; Wang et al., 2018). The human nuclear factor erythroid 2-related factor 2 (NRF2) has been extensively studied for its role as both a sensor of oxidative stress and a positive regulator of antioxidants (Zheng, Gonçalves, et al., 2020). Skn-1 is the C. elegans homolog of Nrf2. In this article, we describe the use of a reporter strain, VP596, for the assessment of oxidative stress. The strain expresses GFP under the control of the promoter of glutathione-S transferase 4 (gst-4), a skn-1 target that is activated upon oxidative stress. In addition, this strain constitutively expresses RFP under the promoter of dop-3, and acts as an internal reference (Leung, Deonarine, Strange, & Choe, 2011; Leung et al., 2013)..

The procedure for neurotoxicity evaluation of nanomaterials detailed here is divided into four Basic Protocols. Basic Protocol 1 describes the synchronization and exposure of the nematodes. Basic Protocol 2 describes a survival assay, which can be used to evaluate acute toxicity of nanomaterials. In addition, this protocol allows users to define the doses to use in the subsequent protocols. Basic Protocol 3describes assays for the evaluation of head thrash and body bend, two locomotion changes that are known to reflect neurotoxicity (Xu et al., 2017). Finally, Basic Protocol 4 describes an assay to assess oxidative stress generated by nanomaterials, using the VP596 transgenic strain.

The aim of this protocol article is to provide a relatively quick method for neurotoxicity evaluation of nanomaterials of interest using C. elegans, and could easily be adopted for researchers without extensive C. elegans experience. The protocols could also be applied for broad-range quick toxicity scanning, and could set the foundation for subsequent mechanistic studies.

STRATEGIC PLANNING:

Before beginning, users should thoroughly characterize their nanomaterials of interest, as this is key for the protocol. This relates to what the nanomaterials consist of (composition and purity) and how they look like (shape and size) (Fadeel, Fornara, Toprak, & Bhattacharya, 2015), and such characterization should be done in the exposure solution for fit-for-purpose. Some of the most commonly characterized features and the corresponding techniques to determine them are listed in Table 1, using cobalt nanoparticles (CoNPs) as an example. Detailed characterization methods can be found in Zheng et al. (2022a).

Table 1.

Commonly characterized features of nanomaterials

Parameter	Values for CoNPs	Method used
Composition (%). Refers to the elements that make the nanomaterial and the coating.	Cobalt Carbon < 8%	Inductively coupled plasma-mass spectrometry (ICP-MS)
Endotoxin contamination. Nanomaterials can be contaminated with lipopolysaccharides (LPS) during production or handling	Under detection limit (0.01 EU/mL)	Tachypleus Amebocyte Lysate (TAL)
Size [nm] average (Min-Max), in water or organic solvent*	In ddH2O: 34.89 (13.34–85.79)	Transmission electron microscopy (TEM)
Z-average [d,nm], in water or organic solvent*	In water: 1404 ± 191.9	Dynamic light scattering (DLS)
Polydispersity, in water or organic solvent*	In water: 0.751	DLS
Zeta potential [mV], in water or organic solvent*	In water: 8.67 ± 3.78	DLS
Ion dissolution, at 2 h, 12 h, and 48 h. Timing depends on exposure duration	24 h: 39.75 ± 1.21 48 h: 45.16 ± 2.50	ICP-MS
Purity	>99%	Trace metal analysis

^*The characteristics in an organic solvent is only needed if the nanomaterial is preferably delivered using the specific solvent.

Next, users should select some initial doses and time courses for exposure. Users can take advantage of populational, experimental, and quantitative structure–activity relationship (QSAR) modeling data (either of the same or a related nanomaterial with similar physicochemical properties) to collect background information of the nanomaterial that could inform an exposure regime. Besides, users are encouraged to perform pilot experiments with a wide range of doses. For example, users can prepare 10-fold serial dilutions from the maximum solubility to 5–10 dilutions. Normally, users should not use doses that result in a survival rate lower than 80%, so as to reduce experimental error.

Lastly, users are advised to prepare concentrated “stock solutions” of nanomaterials for exposure. For CoNPs and other nanomaterials we have examined (TiO₂ and MWCNTs), 5 × concentrated stock solutions are both easy to reconstitute and to handle when using for nematode exposure. For instance, to prepare a 1 mg/mL master solution of CoNPs, weigh 5 mg of CoNPs and reconstitute with 5 mL of ddH₂O in a 15-mL Falcon tube. Sonicate for 10 minutes. Then, prepare 5 × stock solutions of the exposing groups. For example, for final exposing concentrations of 0, 25, 50, and 75 μg/mL CoNPs, prepare four 1-mL tubes of 5 × stock solutions at 0, 125, 250, and 375 μg/mL, using the 1 mg/mL CoNPs master solution and M9 (Table 2).

Table 2.

Preparations for 5 × stock solutions

Exposure doses (μg/mL)	0	25	50	75
Concentrations for 5 × stock solutions (μg/mL)	0	125	250	375
Volume of 1 mg/mL CoNPs (μL)	0	125	250	375
Volume of M9 (μL)	1000	775	750	625

BASIC PROTOCOL 1: Exposure of C. elegans to nanomaterials

In this protocol, we describe the steps to synchronize nematodes and expose them to nanomaterials of interest that have been previously characterized (see Strategic Planning). Here, L1 worms are exposed to various concentrations of the nanomaterials using a liquid exposure method, to keep exposure concentration stable.

If solid exposure is preferred, it is recommended that other protocols be used to evaluate the internal dose of nanomaterials (Cagno et al., 2017; Gonzalez-Moragas et al., 2017), i.e., the true concentration of nanomaterials inside C. elegans, since the nanomaterials are prone to staying in the agar.

Materials:

• Plates with actively growing C. elegans nematodes: N2, wild type (Caenorhabditis Genetic Center, CGC)

• 1 M KPO₄ (see Reagents and Solutions)

• OP50-seeded and unseeded 100-mm NGM plates (see Reagents and Solutions)

• M9 buffer (see Reagents and Solutions)

• ddH₂O (autoclaved)

• 10 M NaOH solution (see Reagents and Solutions)

• Bleach solution (see Reagents and Solutions)

• 30% w/v Sucrose solution (see Reagents and Solutions)

• 5× concentrated stock solutions of nanomaterial-of-interest (see Strategic Planning section for details). If CoNPs is taken as an example, the 5× concentrated stock solutions to use are 0, 125, 250, and 375 μg/mL of CoNPs.

• Incubator (Memmert, IPP260)

• Bench-top centrifuge (Eppendorf, centrifuge 5702)

• Stereomicroscope (Leica, 9i)

• Rotary shaker (IKA, Loopster)

• Vortex (IKA, Vortex 3)

• Ultrasonic cleaner (Fisher Scientific, FS30)

• Centrifuge tubes (Biosharp, BS-150-M)

• Sterile Pasteur pipettes (Nest,318314)

• Microscope slide (Citotest, 188105)

• 1.5-mL Eppendorf tubes (Axygen, MCT-150-C)

Protocol steps:

Synchronization of the nematodes

• 1Use a 200 μl-tip to pick a piece of agar medium with a lot of nematodes at exponential growth phase, and put it up-side-down (i.e. with the worms-side facing OP50) on an OP50-seeded 100-mm NGM plate. This step is also known as “chunking”.

  Chunking three plates is usually enough for exposing the nematodes to four concentrations of nanomaterials. Users can adjust the number of plates or the size of plates for synchronization to suit their purposes.

• 2Incubate the above plates in an incubator at 22 °C.

  This applies to N2 worms. Some transgenic nematodes are maintained at lower temperatures, such as 16 °C. The temperature of the incubator should be adjusted to suit the user’s research aim.

  Plates should be ready for synchronization at Day 2 or 3, depending on the status and strains of nematodes.

• 3Observe plates under the stereomicroscope on Day 2. If the plates are full with gravid adult nematodes, and no or few eggs and hatched larva are present, the plates are at the right stage for the following steps. If not, check again on Day 3.

  If the plates are full of nematodes in mixed stages, users could do a synchronization first (following the same protocol described here until step 20). Although the yield might be low, grow the hatched larval nematodes on one OP50-seeded NGM plate until exponential growth phase and repeat the protocol.

• 4Once ready, rinse the nematodes off the plates twice with approx. 3 mL of M9, and collect the suspension of all three plates to a single 15-mL centrifuge tube.

  The plates can then be checked under the stereomicroscope to confirm that the majority of nematodes have been washed off. If more plates are used, the suspension can be collected in multiple centrifuge tubes.

• 5Vortex the tubes thoroughly and centrifuge them at 1800 rpm for 1 minute at room temperature.
• 6Discard the supernatant and fill the tube with M9.
• 7Repeat step 5–6 until the supernatant is clean and clear.
• 8Remove the supernatant and add 5 mL of bleaching solution to the worm pellets.

  If the worm pellet is larger than 1 mL, more bleaching solution should be added, maintaining a 1 mL pellet: 5 mL bleaching solution ratio.

• 9Leave the centrifuge tubes on a rotary shaker for 5 minutes and vortex for 10 seconds every minute.
• 10After 5 minutes, observe nematodes under the microscope and make sure all eggs are released from the nematodes.

  If there are still lots of intact nematodes after 5 min, extend the bleaching time or replace with new bleach solution.

• 11Add M9 to dilute bleach, vortex, and centrifuge at 2800 rpm for 1 minute at room temperature.
• 12Remove all liquid with a pipette (manually or with a vacuum machine) and fill with M9. Vortex and then centrifuge at 1800 rpm for 1 minute at room temperature. Repeat the wash twice, for a total of three washes.

  Always change pipette tips in between, to avoid contamination.

• 13Remove liquid and add 10 mL of 30% w/v sucrose solution. Vortex and mix well.

  Proper mixing is needed to ensure success of the gradient centrifugation.

• 14Centrifuge at 700 rpm for 8 minutes with gradual deceleration.
• 15Collect eggs from the top layer (no more than 3 mL for a 15-mL tube), and place them in a new 15-mL tube.
• 16Add 10 mL of ddH₂O to dilute the eggs.
• 17Vortex and centrifuge at 2700 rpm for 1 minute.
• 18Remove the supernatant, add 1 mL of M9, and mix well by gently pipetting up-and-down.
• 19Pipette all the eggs onto unseeded NGM plates.
• 20Allow plate to dry at room temperature and place in the 22 °C incubator for at least 18 h (depending on the growth rate of the strain) to obtain larva stage 1 (L1) nematodes.

Exposure

• 21Wash the plate with M9 to collect the synchronized L1 nematodes into a 15-mL tube.
• 22Determine the number of nematodes per μL. First, add M9 to the tube to 10 mL. Mix the nematodes and take 2 μL onto a clean microscope slide. Repeat for a total of three separate aliquots. Count the number of nematodes in each 2 μL-drop under the stereomicroscope. Divide the number of 2 μL-drop by 2 to get the number of nematodes per μl for each drop. Then, average the three numbers to get the estimated number of nematodes per μl in the 10 mL solution.

  Make sure to mix the nematodes each time immediately before taking out each 2 μL-aliquot, to obtain an accurate estimate of the concentration of nematodes in the tube.

• 23Make the final concentration of nematodes to 10 nematodes per μL by either concentrating or diluting.

  In this protocol, L1 nematodes are used. If L4 or young adult nematodes are to be exposed to suit the user’s goal, normally 2–5 nematodes per μL is enough.

• 24Sonicate the 5 × nanomaterial stock solutions (0, 125, 250 and 375 μg/mL of CoNPs) for 10 min in an ultrasonic cleaner. For each exposure group, take 400 μL of the worm suspension to a 1.5-mL microcentrifuge tube and then add 100 μL of sonicated nanomaterial stock solution to the nematodes.

  Sonicate the stock solution immediately before every exposure. In this step, the 5 × stock solutions will be diluted to their final concentrations. Here, the nematodes were exposed to CoNPs at final concentrations of 0, 25, 50, and 75 μg/mL, i.e., control and three exposure groups. If the nanomaterial of interest is being studied with nematodes for the first time, pilot experiments using a wider range of exposure doses (e.g. 10-fold serial dilution) and/or exposure times are recommended.

• 25Incubate nematodes with the nanomaterial for 12 h on a rotary shaker.

  The exposure duration can be shortened, if needed, for acute exposure. If chronic exposure is preferred, inactivated bacterial food needs to be supplemented to the nematodes.

• 26Centrifuge at 3000 rpm for 1 min, discard the solution, and resuspend the nematodes in 100 μL M9 buffer. Plate the exposed nematodes on NGM plates with OP50 and let them recover by incubating the plate for an extra 2 h in the incubator at 22 °C.

  For nanomaterials that are prone to aggregate, nematodes should be spread on a “transit” NGM plate first, and then manually transferred using a platinum picker to the recovery plate.

• 27Proceed to analyze survival (Basic Protocol 2), locomotive behavior (Basic Protocol 3), and/or oxidative stress (Basic Protocol 4).

BASIC PROTOCOL 2: Survival assessment

Survival is a direct indicator of a nanomaterials’ acute toxicity. It is also informative in dose selection for behavioral (Basic Protocol 3) or mechanistic (Basic Protocol 4) studies. Here, the exposed nematodes from Basic Protocol 1 are used directly for a survival assay. Briefly, the nematodes are placed in newly prepared NGM plates, and the living nematodes are then counted on day 3. The survival rate is then calculated for each exposure group and the control.

Materials:

• Control and exposed C. elegans, from Basic Protocol 1

• Unseeded 35-mm NGM plates (see Reagents and Solutions)

• M9 buffer (see Reagents and Solutions)

• ddH₂O (autoclaved)

• Overnight E. coli OP50 liquid culture (CGC) in Lysogeny Broth (LB, Gibco, 10855001) or similar.

• Safety cabinet with UV (Heal Force, HFsafe-1200LC)

• Incubator (Memmert, IPP260)

• Stereomicroscope (Leica, 9i)

• Vortex (IKA, Vortex 3)

• Sterile Pasteur pipettes (Nest,318314)

• Platinum picker

Protocol steps:

1. Label an adequate amount of 35-mm unseeded NGM plates with exposure dose, number of replicates, and date.

   The required number of plates equals the number of groups (different exposure doses and controls) × 3 (technical triplicates), plus 3 extra plates. For example, here, to test the survival of nematodes exposed to 0, 25, 50 and 75 μg/mL of CoNPs, users would need (4 × 3) + 3 = 15 plates.

2. Seed a drop of an overnight culture of OP50 (approx. 20 μL) onto each 35-mm plate. Sterilize the OP50 using the UV light from a safety cabinet for 30 min.

   After UV treatment, the OP50 will be inactivated and observed as a very thin layer, resulting in easy subsequent observation of nematodes.

3. Transfer approx. 30–40 nematodes (from Basic Protocol 1) per plate to the corresponding freshly prepared 35-mm plates from step 2. Record the number of nematodes per plate.

4. Two days later, score the nematodes with a stereomicroscope as alive or dead. Dead nematodes can be confirmed by touching the head region with the point of a picker.

   Sometimes, after exposing to nanomaterials, users may observe abnormally short or thin nematodes, or nematodes at early developmental stages, which may indicate developmental toxicity of the nanomaterial. If this happens at a regular basis (often and to a large fraction of the population), users are encouraged to examine developmental neurotoxicity, such as brood size, embryo hatchability, and generation time (Lu, Bu, Ma, & Liu, 2020).

5. For each group, calculate the survival rate by dividing the number of alive nematodes by the total number of nematodes, and display it as a percentage. Calculate the average among the triplicates in each group and plot the graph with a box plot to see data distribution. Repeat Basic Protocols 1 and 2 three times to get biological replicates (i.e., perform three individual experiments).

   See Figure 1 and the Understanding Results section for sample data.

Figure 1. CoNPs exposure induces lethality in C. elegans.

WT L1 C. elegans nematodes were exposed to 0, 25, 50, and 75 μg/mL of CoNPs for 12 h in the absence of food, as described in Basic Protocol 1. The nematodes were then scored for lethality, as described in Basic Protocol 2. The experiment was performed with 3 technical replicates, with 40 animals per plate. The experiments were independently repeated twice. The data are presented in a box plot showing % of survival, compared to the control. * P < 0.05 and ** P < 0.01 compared with control group using one way ANOVA and Dunnett’s multiple comparisons test.

BASIC PROTOCOL 3: Assessment of locomotion behavior

Here, users will assess locomotion behavior using the exposed nematodes from Basic Protocol 1. Users will evaluate the two most commonly used endpoints to assess locomotion behavior, namely, head thrash and body bend. Briefly, users will manually pick exposed or control nematodes to new unseeded NGM plates. After the nematodes recover from exposure, users will record videos of each plate and analyze the videos for body bend and head thrash to evaluate locomotion behavior upon nanomaterial exposure.

Materials:

• Control and exposed C. elegans from Basic Protocol 1

• Unseeded NGM plate (see Reagents and Solutions)

• Digital stereomicroscope coupled to a computer (Leica, S9i)

• Platinum picker

Protocol steps:

1. For each exposure or control group, transfer approx. 40 nematodes from the recover plate (Basic Protocol 1) to a new, unseeded NGM plate, with a platinum picker.

2. Let the nematodes recover for 1 min in the NGM plates so as to become accustomed to the environment. At the same time, adjust the magnification of a stereomicroscope such that the entire plate is right inside the field-of-view (neither too small, to be able to observe the movements of nematodes, nor too big, to be able to track all nematodes).

   The magnification should remain constant among the plates for examination.

   Users are recommended to recover nematodes (both control and exposed plates) one-by-one under the stereomicroscope with its light on. Then, the plates can be recorded directly in the next step.

3. Record each plate with nematodes using the camara from a digital stereomicroscope for 70 seconds. Save the recording files to the computer.

   Sometimes the start and end of the recording can exhibit some delay caused by the communications between the camera and the recording software. Therefore, 70 seconds of recording is suggested here, to ensure the recorded video is more than 60 seconds.

4. Count head thrashes of each nematode for 1 min from the recorded videos. Head thrash is defined as a change in the direction of bending at the mid body (see Figure 2A).

5. Count body bend of each nematode for 30 seconds from the beginning of the recorded videos. Body bend is defined a change in the direction of the worm corresponding to the posterior bulb of the pharyngeal along the y axis, assuming the worm was travelling along the x axis (see Figure 2B).

   To minimize potential artifacts, it is highly recommended to follow the above protocols, i.e., record the locomotion of nematodes and then count the movements per nematode from the video. Alternatively, the movements can be recorded manually with the help of a counter and a timer.

6. Plot the graph as a box plot to see data distribution. Repeat Basic Protocols 1 and 2 three times, to obtain biological replicates.

   See Figure 3 and Understanding Results section for sample data.

Figure 2. An illustration of locomotion behavior.

(A) One complete head thrash is scored when the head moves to one side and back. (B) One complete body bend is scored when there is a change in the direction of the worm with respect to the posterior bulb of the pharyngeal along the y axis, assuming the worm was travelling along the x axis. In this diagram, 3 body bends are displayed.

Figure 3. CoNPs impairs locomotion in C. elegans.

WT L1 C. elegans nematodes were exposed to 0, 25, 50, and 75 μg/mL of CoNPs for 12 h in the absence of food. Locomotion behavior was then analyzed. (A) head thrash and (B) body bend numbers were both impaired in response to CoNPs, compared to control. 40 animals were analyzed per group. ** P < 0.01 and *** P < 0.001 compared with control group using one way ANOVA and Dunnett’s multiple comparisons test.

BASIC PROTOCOL 4: Analysis of oxidative stress

As mentioned in the introduction, oxidative stress is one of the key mechanisms for nanomaterials-induced toxicity. Here, we take advantage of a fluorescent transgenic reporter strain, VP596, which expresses both GFP and RFP, under the control of the promoter for the skn-1 target glutathione-S transferase 4 (gst-4) and under the constitutive promoter dop-3 (Leung et al., 2011), respectively. Skn-1 is the C. elegans homologue of Nrf2, a universal redox inducer and regulator. Here, users will expose these nematodes to the nanomaterial of interest (following Basic Protocol 1) and will then proceed to evaluate GFP and RFP intensities using a plate reader. By measuring GFP fluorescence intensity upon nanomaterial exposure, the generation of oxidative stress can thus be indirectly detected. Moreover, for quantitative or high-throughput analysis, GFP intensity can normalized to that of RFP, which is stably expressed in the VP596 strain, so that the GFP can be standardized by number of worms. Of note, these transgenic nematodes are known to react similarly to wildtype to oxidative stress (Detienne, Van de Walle, De Haes, Schoofs, & Temmerman, 2016; Kittimongkolsuk et al., 2021; Kotlar et al., 2018).

Materials:

• C. elegans strain VP596 (dvIs19 [(pAF15) gst-4p::GFP::NLS] III. Oxidative stress-inducible GFP. vsIs33 [dop-3::RFP] V.) (Caenorhabditis Genetic Center, CGC)

• 5X nanomaterial stock solutions (see Strategic Planning).

• Fluorescent plate reader (FluoStar OPTIMA, BMG LabTech)

• Sterile Pasteur pipettes (Nest,318314)

• 96-well black plates (Corning, 07-200-590)

• 1.5-mL tubes

Protocol steps:

1. Grow and expose VP596 nematodes to the nanomaterial of interest (and control), following Basic Protocol 1, Steps 1 to 26.

2. Prepare serial nanomaterial solutions of the test concentrations using the 5X nanomaterial stock solutions using M9 buffer in a 1:4 ratio. Transfer 100 μL of each of the serial nanomaterial solutions to a 96-well black plate in duplicates, to serve as negative controls wells (See Figure 4, in blue).

   This step is to prepare negative control wells so as obtain the background fluorescent values of nanomaterials with different concentrations at excitation/emission 544/ 590 nm (RFP) and GFP: 485 /520 nm (GFP).

3. Wash the exposed/control plates from step 26 in Basic Protocol 1 and make the final concentration of nematodes to be 10 nematodes per μL, using the same method from Basic Protocol 1 Step 22 and 23.

4. Transfer 100 μL of the nematodes to the 96-well black plate in triplicates. This will result in approx. 1000 nematodes per well (see Figure 4, orange).

   The number of nematodes to use in each well is determined by the detection limit of the plate reader.

5. For each well, measure levels of RFP and GFP florescence at excitation/emission 544/ 590 nm and 485 /520 nm, respectively.

6. Divide GFP florescence values by RFP florescence values of each well to normalize the data to worm number. Perform the appropriate statistical tests and plot using box plots.

   See Figure 5 and Understanding Results section for sample data.

Figure 4. Diagram of the 96-well plate used for the examination of oxidative stress.

The background fluorescence levels of nanomaterials are determined by adding different concentrations in duplicates in separate wells (blue). Fluorescence of exposed nematodes are in evaluated in triplicates (orange). In this example, worms were exposed to 0, 25, 50, and 75 μg/mL CoNPs.

Figure 5. CoNPs induces oxidative stress in C. elegans.

VP596 L1 C elegans nematodes (with oxidative stress inducible gst-4p::GFP and stably expressed dop-3::RFP) were exposed to CoNPS at 0, 25, 50, and 75 μg/mL for 12 h in the absence of food. GFP and RFP signals were then measured with a fluorescent microplate reader. The experiment was independently performed twice, each with 3 technical replicates with 1000 animals per well. Oxidative stress levels (using GFP intensity as a proxy) were standardized by number of nematodes using RFP intensity. The GFP/RFP ratio of control group (treated with 0 μg/mL of CoNPs) was set to 1. * P < 0.05, *** P < 0.001 compared with control group using one way ANOVA and Dunnett’s multiple comparisons test.

REAGENTS AND SOLUTIONS:

1 M KPO₄ (1L solution)

• 108.3 g KH₂PO₄ (Sigma P5655)

• 35.6 g K₂HPO₄ (Sigma P0662)

• Dissolve with ddH₂O to a final volume of 1000 mL

• Calibrate pH to 6.0 with KOH

• Filter the prepared reagent into an autoclaved bottle using a 0.22 μm sterile filter

10 M NaOH

• Weigh 20.0 g of NaOH (Macklin, S817970–500)

• Dissolve with ddH₂O to 50 mL.

• Stir to cool.

Bleach Solution

• 2 mL of bleach

• 0.5 mL of 10 M NaOH

• 7.5 mL of ddH₂O in a 15-mL Falcon tube covered with aluminum foil.

• Mix thoroughly.

• Prepare fresh right before use.

NGM plates

• 3 g NaCl (Shanghai Hushi, 10019308 OR Sigma, S9888)

• 17 g Agar (Guangzhou Saiguo biotech, Biofroxx, 8211GR500 OR Sigma, 05039)

• 2.5 g Peptone (Shanghai Yuanye, S26075 OR Sigma, P5905)

• 975 mL ddH₂O

• Autoclave for 45 min on liquid cycle. Cool to 50°C.

• Add the following components:

  • 1 mL of 5 mg/mL Cholesterol (Shanghai Yuanye, S11040-100 OR Sigma, C8667)
  • 1 mL of 1 M CaCl₂ (Fisher Scientific, 349610250 OR Sigma, C1016)
  • 1 mL of 1M MgSO₄ (Xilong huagong Chemical OR Sigma, M7506)
  • 25 mL of 1M KPO₄ (see recipe)
  • 0.5 mL of Streptomycin sulfate (Shanghai Yuanye, S17058-25 OR Millipore, 516104-M)

• Pour into 100-mm or 35-mm plates.

  A small container (e.g. a beaker) or sterile pipettes can be used to distribute medium. Gently swirl the medium to avoid solidification. Also, the medium can be kept in a 50°C water bath before or while in use.

• If OP50 seeded plates are needed: Spread 200 μL of an overnight-grown culture of OP50 E. coli (CGC) on the above prepared 100-mm NGM plates and leave them at room temperature overnight.

• Store at 4 °C refrigerator for up to 2 months.

• Preparation should be conducted with sterile technique.

M9 buffer

• 6 g KH₂PO₄ (Macklin, P815662–500)

• 12 g Na₂HPO₄ (Macklin, S818100–500)

• 10 g NaCl (Shanghai Hushi, 10019308 OR Sigma, S9888)

• ddH₂O up to 2 L

• Autoclave for 45 min on liquid cycle.

• Cool to 40 °C, and add 2 mL of 1M MgSO₄ (Xilong huagong Chemical OR Sigma, M7506)

• Store at room temperature for up to 6 months.

• Preparation should be conducted with sterile technique.

• It is suggested to use M9 buffer in a working 50-mL conical for easier handling.

Sucrose (30% w/v)

• Weigh 240 g of Sucrose (Guangzhou Saiguo biotech, Biofroxx, 1245GR500)

• Add ddH₂O to 800 mL.

• Autoclave.

COMMENTARY:

Background Information:

Nanomaterials have been extensively used in medical, industrial, and environmental settings. The accumulation of nanomaterials can cause potential adverse effects to human health, including neurotoxicity, neuroinflammation, and neurodegeneration (Boyes & van Thriel, 2020; Migliore, Uboldi, Di Bucchianico, & Coppedè, 2015). Indeed, numerous studies have reported the neurotoxicity of nanomaterials. For instance, exposure to graphene oxide nanomaterials and polystyrene nanoparticles have been shown to affect survival or lifespan in C. elegans (Kim, Eom, Choi, Hong, & Choi, 2020; Shang et al., 2021). In addition, induction of oxidative stress and generation of reactive oxygen species have been reported in response to graphene-based nanomaterials, cadmium telluride quantum dots, multi-walled carbon nanotubes, TiO₂ nanoparticles, polystyrene nanoparticles, and silver nanoparticles (Kim et al., 2020; Shang et al., 2021; Q. Wu et al., 2013; Q. Wu et al., 2012; T. Wu et al., 2015; Zhang et al., 2021). Moreover, graphene-based nanomaterials, cadmium telluride quantum dots, silver nanoparticles, and aluminon nanoparticles have been shown to decrease the number of neurotransmitters and receptors (Kim et al., 2020; Y. Li, Yu, Wu, Tang, & Wang, 2013; T. Wu et al., 2015; Zhang et al., 2021). Similarly, impairments of various neurons (e.g., AFD sensory neurons, RMEs motor neurons, dopaminergic neurons) have been observed in response to graphene-based nanomaterials, silver nanoparticles, copper oxide nanoparticles, and cadmium telluride quantum dots (Kim et al., 2020; Mashock et al., 2016; Piechulek & von Mikecz, 2018; Zhao, Wang, Wu, Li, & Wang, 2015). In addition, behavioral defects, including alterations to body bending, head thrashing, pharyngeal pumping, and defecation intervals, have been reported upon exposure to graphene-based nanomaterials, cadmium telluride quantum dots, TiO₂ nanoparticles, silica nanoparticles, polystyrene nanoparticles (Kim et al., 2020; Scharf, Gührs, & von Mikecz, 2016; Shang et al., 2021; T. Wu et al., 2020). Last but not least, nanomaterials such as silica, silver, or other airborne nanoparticles could induce neurodegenerative damage, such as β amyloid formation and protein aggregation (Piechulek & von Mikecz, 2018; Scharf et al., 2016; von Mikecz & Schikowski, 2020).

C. elegans has been extensively used in neurotoxicity evaluations due to its many advantages as a model system. These evaluations typically include lethality, behavioral, and molecular endpoints (Lawes et al., 2020; Natale et al., 2020; Ruszkiewicz et al., 2018). Here, we describe how to quickly examine the neurotoxicity of test nanomaterials using C. elegans by evaluating survival, locomotion, and oxidative stress, which are standard endpoints in C. elegans toxicology assays, using a liquid exposure regime. Instead of using a single endpoint, three endpoints were chosen here to attempt to study the potential neurotoxicity of a nanomaterial of interest more comprehensively. Importantly, in the procedure described here, the exposure of nanomaterials is performed in the absence of OP50, to reduce potential artifacts. This is important, because some engineered nanoparticles with positively charged coating have been shown to exhibit a false positive toxic effect when they agglomerate with E. coli bacteria (Hanna et al., 2018).

The set of protocols described here could readily be scaled-up for fast screening of test nanomaterials’ neurotoxicity. On the other hand, the protocols here could be used as the first step to find sub-lethal dose ranges to unveil the mechanisms of nanomaterial-induced neurotoxicity.

Critical Parameters:

When performing synchronization, the stage of nematodes is critical for the success of this procedure. If the number of gravid nematodes is low, only a few eggs will be collected, which will then lead to insufficient number of larval nematodes for exposure and the subsequent assays. Thus, we suggest to observe the “parent” plates closely, and “chunk” sufficient larval nematodes (approx. 300–400 eggs or L1 nematodes or 20 gravid adults) to fresh OP50-seeded NGM plates. Moreover, when the plates are incubating, check at least twice a day on day 2 and day 3, in order to proceed to synchronization at a good time (i.e. when lots of gravid nematodes are visible, with no or few eggs and no larval nematodes). In addition, the bleaching step (lysis of nematodes and release of eggs) and sucrose centrifugation steps are key to yield sufficient number of larvae. Therefore, we strongly encourage proper mixing of nematodes (bleaching step) or lysates (sucrose step) with reagents in both steps.

During exposure, the handling and storage of nanomaterials could significantly affect the results. For example, users must have a clear idea of how many ions have been released from the metal nanomaterials and how that contributes to neurotoxicity, from the characterization of nanomaterials. This may significantly interfere with the results or data interpretation. Besides, exposure to light (under laboratory lighting or sunlight) and slight changes in temperature have been demonstrated to influence the cellular uptake of engineered nanoparticles and, in turn, their induced toxicity (L. L. Maurer, I. T. Ryde, X. Yang, & J. N. Meyer, 2015). Thus, such conditions need to be monitored and standardized. Moreover, the stock solutions of the nanomaterial of interest need to be sonicated thoroughly to homogenize the solution prior to exposure. For some nanomaterials, a thorough vortex is also recommended.

For the assessment of locomotion behavior, the key step is to count the number of body bend and head thrash. It is difficult to count the movements by eye, and it is hard to track the moving worms. Therefore, recording videos of nematodes is highly recommended. The videos can then be played at different speeds and replayed as many times as necessary for counting and re-examining. Moreover, in the protocol, we suggest users to record a time range (70 seconds) that is longer than the counting time frame (30 and 60 seconds), so as to avoid incomplete recording of the videos due to the delay between software and camara.

When carrying out the oxidative stress analysis, it is important to pay attention to the GFP and RFP fluorescent readings. First of all, they need to be in the detection range, so as to produce a reliable result. For the plate reader we routinely use, about 1000 nematodes per well are recommended for larval stage 1 (L1) nematodes, and 200 of L4 or adult nematodes. A pilot experiment with serial dilution of worms could be carried out first to check the fluorescent detection range. In such way, users can adjust number of nematodes per well by fluorescence signal. Moreover, potential artifacts by autofluorescence of nanomaterials need to be excluded. As stated in Basic Protocol 4, step 2, a series of nanomaterial exposing solutions have to be prepared and examined for fluorescence.

Troubleshooting:

Table 3 describes some common problems with the protocols described, along with the possible causes and solutions.

Table 3.

Troubleshooting guide for neurotoxicity evaluation induced by nanomaterials.

Problem	Possible Cause	Solution
No or few eggs collected after sucrose gradient centrifugation (Basic Protocol 1).	Insufficient release of eggs	Extend the bleaching treatment for 3 more minutes
	The nematodes were at the wrong stage.	“Chunk” plates a day later, in addition to day 1 (Step 1, BP1).
The total number of nematodes (both alive and dead) on the plate is lower than that two days ago (Basic Protocol 2).	Nematodes were dried up in the inner wall of the NGM plate.	Check the inner wall of the NGM Petri dish. Maintain proper humidity conditions.
Locomotion behavior data has a large standard deviation (Basic Protocol 3).	There might be one or two “outliers” in the data pool.	When observing the nematodes, leave out the nematodes moving aberrantly fast or slow (e.g. > 2 fold or < 0.5 fold than the average speed in the same exposure group).
	The sample number is too low.	Increase sample number.
No difference in GFP/RFP signals between groups (Basic Protocol 4).	Dose too low/ exposure time too short	Adjust exposure conditions: extend exposure duration and/or incubate with nanomaterials at higher doses
	Signal below or above the detection limit of the plate reader.	Adjust the number of nematodes in each well until the fluorescent signals of 100, 500, 1000 and 5000 worms are linear.
	Autofluorescence from nanomaterials interferes with the signal.	Check the negative control wells from BP4 step 2 and see whether fluorescent signal is detectable in the serial dilution of nanomaterials.
	The nanomaterial tested at the specific dose induces ROS but does not activate skn-1-dependent pathways.	Examine with other assays, such as H2DCFHDA fluorescent probe (Yoon, Lee, & Cha, 2018; Zheng, Chen, et al., 2020), to verify whether oxidative stress is induced by the nanomaterial.

Understanding Results:

Below we describe some sample data for tests we have conducted with cobalt nanoparticles (CoNPs). Wildtype nematodes were exposed to CoNPs for 12 h and the results are shown in Figure 1, as a percentage. The selection of doses and exposure times was based on our pilot experiments. CoNPs induce a clear decrease in the percentage of surviving nematodes. The result suggests that CoNPs cause acute toxicity to L1 nematodes, starting from a concentration of 25 μg/mL.

We further subjected N2 L1 nematodes to CoNPs using the same exposure conditions as above, for behavioral assays. Times of head thrash in 1 min (Figure 3A) and body bend in 30 s (Figure 3B) are shown. Both the times of head thrash and body bend were reduced upon CoNPs exposure, suggesting that CoNPs induces alteration of locomotion behaviors.

To assess oxidative stress, VP596 nematodes were exposed to CoNPs using the same exposure conditions as in Figures 1 and 3. Increased activity of SKN-1, the Nrf2 homolog, and expression of its target genes (in this case, gst-4), is demonstrated as an increase in the GFP/RFP ratio in the CoNPs-exposed groups (Figure 5), in a dose-dependent manner. For better comparison, the ratio of control group was set to 1. Data were then examined using the same statistical analysis methods as survival assay. It is noteworthy that in this assay, skn-1-dependent GFP expression is used as a proxy for an increase in ROS. It should be noted that even in the absence of GFP upregulation in response to a test nanomaterial, oxidative stress could still be triggered by the test material, but in a way that is skn-1 independent (such that it would not result in higher GFP levels). Of course, it is also possible that the nanomaterial does not trigger ROS at all. Similarly, upregulation of GFP does not necessarily mean an increase in ROS, as the gst-4 promoter may respond to other signals as well. Therefore, these elements should be taken into account in the interpretation of the results. If no differences are observed, it is possible that the nanomaterial tested does not result in oxidative stress, or one that can be evaluated via skn-1-dependent pathways. Other than that, check Table 3 for possible causes and solutions.

Time Considerations:

In total, Basic Protocol 1 will take approx. four days. It will take 0.5 h to chunk plates, two to three days for the nematodes to grow, 2 h for synchronization, 18 h for the nematodes to hatch and reach L1 stage, 2 h for setting up the exposure, and 12 h for exposure.

For Basic Protocol 2, the preparation of 35-mmplates for survival will take 1 h, which can be performed at the last hour of nematode exposure, to save some time. The transfer of exposed nematodes (for 4 group) will take 2 h for experienced users. The transferred nematodes are then allowed to grow for two days. After that, it will take 1 h to score the nematodes as alive or dead and another 1 h for data analysis. Thus, Basic Protocol 2 will take three days to perform.

For Basic Protocol 3, it will take 2 h to carry out the experiments and record the video. The counting of the movements from the video clips depends on the number of nematodes. Roughly, with practice, it will take 1 h to count the locomotion behavior of 40 nematodes. The data analysis will take another 1 hour. In summary, Basic Protocol 3 will take ~7 hours.

Basic Protocol 4 will take 2 h. It will take 1 h to set up the 96-well plate and read the plates, followed by another 1 hour for data analysis.

DATA AVAILABILITY STATEMENT:

The data that support the findings of this study are available from the corresponding author upon reasonable request.