Characteristics of CNFs and CNCs
The size distribution of CNFs and CNCs was assayed (Fig. 1A and B). The diameter of the CNFs mainly distributed between 50 nm and 70 nm, while the diameter of CNCs mainly distributed in the range of 24–35 nm. SEM images showed that CNFs and CNCs were capable of dispersing on mica substrate without tendency for agglomerating (Fig. 1C and D). It should be mentioned that any surface aggregation of the nanoparticles was not observed. The shape of CNFs is mainly long fibrous, with the length between 1 ~ 2 µm and diameter 15–40 µm, whose aspect ratio varies from 15 to 40. With a rod-shaped structure, the CNCs length is between 250–400 nm, aspect ratio in 10–11.
Toxicities of CNFs and CNCs on zebrafish embryos at the whole animal level
To assess the potential toxicity of CNs, we treated the wild type zebrafish embryos with CNFs or CNCs during 8–96 hpf. The toxicological responses such as mortality, hatching, and abnormality were observed and recorded (Fig. 2). At the concentration between 30 mg/ml and 0.1 µg/ml, CNFs and CNCs showed no dosage-dependent toxicological responses in treated zebrafish embryos, respectively at 48, 72, and 96 hpf.
Enrichment Of Cns In Zebrafish Larvae
According to the scheme in Fig. 3A, the wild type zebrafish larvae were treated with FITC labelled CNCs (CNC-FITC). Fluorescence microscope imaging indicated that CNC-FITC was enriched in the whole embryo at both 10 µg/ml and 1 µg/ml (Fig. 3B). The enrichment of CNC-FITC also at both 10 µg/ml and 1 µg/ml was further observed by confocal microscope (Fig. 3C). FITC coupled CNCs treatments showed that CNs could be enriched in zebrafish larvae.
Cnfs Obviously Injured The Motor Ability
After treated with CNFs or CNCs, the motor ability of zebrafish larvae receded obviously. Thus, we carried out locomotion analyses in order to quantitatively obtain the injury of CNs. Here, we showed that CNFs of 30 mg/ml obviously inhibited the motor ability of zebrafish larvae through recording the swimming track (Fig. 4A) and retention time displayed with heat map (Fig. 4B). In addition, the average swimming velocities (Fig. 4C) and distances (Fig. 4D) in every minute were obviously decreased by CNFs treatments. Similarly, compared with control group, the average swimming accelerations (Fig. 4E) and decelerations (Fig. 4F) in every minute were obviously decreased by CNFs treatments. Moreover, we found CNF treatments significantly increased alteration of swimming direction through recording the angular velocity (Fig. 4G) and turning angle (Fig. 4H). These data demonstrated that CNF exposures significantly injured zebrafish larva motor ability.
Cnfs And Cncs Significantly Inhibited Motor Neuron Development
Judged from locomotion analyses, we deduced there must be some wrong with the nervous system in the CNs treated zebrafish larvae. So, we investigated the effects of CNs on the trunk motor neurons using transgenic zebrafish line Tg(Hb9:GFP::Kdrl:ras-mcherry). Meanwhile, we detected the effects of CNs on the zebrafish trunk blood vessels (Fig. 5A). Comparing with control group, the number of CaP motor neurons and the length of their projections in the zebrafish larvae treated with 30 mg/ml of CNFs or CNCs were significantly reduced (Fig. 5B). In addition, the projections of these motor neurons were disorganized (Fig. 5B). While the blood vessels showed no obvious developmental defects in the CN treated zebrafish larvae (Fig. 5B). By calculating the missing percent of CaP motor neurons, both 30 mg/ml of CNFs and CNCs significantly resulted in the CaP motor neuron missing in the trunk of zebrafish larvae (Fig. 5C). Moreover, almost all CaP motor neurons showed developmental defects. And comparing with control group, the RoP and MiP motor neuron development was also obviously abnormal. These results suggest the motor neuron differentiation and morphogenesis was interrupted.
mRNA-seq analyses revealed CNs directly injured nervous system.
The injured swimming ability and motor neuron phenotype of CNs treated zebrafish larvae directed us to focus on the neurotoxicity. Here, we investigated the molecular alterations using mRNA sequencing. GO enrichment analyses demonstrated that CN treatments participated in the nervous system injury, including axon guidance, neuron projection morphogenesis, spinal cord motor neuron differentiation, axon extension, axonogenesis, synaptic signaling, and etc. biological processes (Fig. 6A). In addition, cellular component enrichment and molecular function enrichment analyses revealed that the different expression genes well matched with the regulated biological processes (Figure S1 and 2). The signaling pathway enrichment analyses showed that CN treatments acted on the multiple typical signaling pathways in neurons, such as calcium signaling pathway, neuroactive ligand-receptor interaction, MAPK signaling pathway, Notch signaling pathway, Wnt signaling pathway, regulation of actin cytoskeleton, and etc. (Fig. 6B).