Respirable α-Quartz Silica Triggers Immune–Inflammatory–Fibrotic Initiation in Zebrafish Embryos via Hindbrain Ventricle Microinjection: Implications for Silicosis Early Risk Assessment

TL;DR

This study shows that respirable silica can trigger early signs of silicosis in zebrafish embryos, offering a new model for assessing silica-related health risks.

Contribution

The study introduces zebrafish embryos as a novel model for early silicosis risk assessment using respirable α-quartz silica.

Findings

Respirable α-quartz silica caused decreased survival and increased malformation rates in zebrafish embryos.

Exposure led to immune and fibrotic gene upregulation and behavioral changes in embryos.

Early inflammatory and fibrotic responses were observed without mature fibrosis.

Abstract

Silicosis, an irreversible occupational lung disease resulting from prolonged exposure to respirable crystalline silica, faces challenges due to limitations in existing mammalian models. This study evaluated whether laboratory-prepared respirable α-quartz silica could induce immune cell–inflammatory–fibrotic initiation related to silicosis in zebrafish embryos as a tool for early toxicity assessment. Zebrafish embryos at 48 h post-fertilization (hpf) were microinjected into hindbrain ventricle with respirable α-quartz silica (test material 3.056 μm vs. standard material 3.217 μm) derived from natural α-quartz ore. The results indicated a significant decrease in zebrafish survival rates and an increase in malformation rates following exposure respirable α-quartz silica materials. Additionally, alterations in midbrain and hindbrain lengths were observed, while body length remained unaffected. Behavioral assessments revealed reduced touch response rates, decreased average speed, and less time spent in the central zone during open field tests in the treatment groups. In vivo imaging demonstrated sequential recruitment of neutrophils (peak at 18 h post-injection) and macrophages (peak at 24 h post-injection). qPCR analysis revealed upregulation of inflammation-related genes (tnf-α, il-6, il-1β) and fibrosis-related genes (tgf-β, acta-2, collagen). Moreover, the hydroxyproline content, a marker for fibrosis, was significantly elevated, although no mature fibrosis was observed histologically. These findings demonstrate that respirable α-quartz silica elicits pathophysiological changes associated with silicosis early initiation in zebrafish embryos. This supports the utility of the zebrafish embryo as a practical tool for early toxicity assessment and mechanistic studies of silica-induced immune–inflammatory–fibrotic initiation, with potential implications for silica exposure early risk warning.

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1. Introduction

Silicosis is an irreversible occupational lung disease caused by chronic exposure to crystalline silica (SiO_2_) dust. This exposure leads to persistent pulmonary inflammation, the formation of silicosis nodules, and pulmonary fibrosis. Severe cases can result in respiratory failure and death, with an annual global mortality exceeding 12,000, and incidence rates are increasing in developing countries [1,2]. α-quartz, a highly pathogenic crystalline form of free silica with particles smaller than 10 μm, can bypass respiratory defenses, accumulate in alveoli, and be engulfed by macrophages, triggering a cascade of pathological responses through lysosomal membrane disruption and activation of inflammatory pathways [3,4,5,6]. It is worth noting that the early inflammatory response induced by silica exposure is identified as the critical initiating event for subsequent irreversible pulmonary fibrosis, making it a key target for silicosis early risk assessment and intervention. Despite extensive research efforts, effective treatments for reversing lung damage in silicosis remain elusive. The primary challenge lies in the inadequacy of current animal models to accurately replicate disease progression in humans. These models are costly to maintain, require prolonged experimental periods, and are not conducive for high-throughput screening.

Traditional silicosis research predominantly relies on mammalian models such as mice and rats [7,8,9,10]. However, these models do not fully replicate human pathological features and do not allow for real-time observation of inflammation–fibrosis dynamic changes. This limitation arises from structural differences in the lungs of these mammalian models compared to those of humans and the lack of in vivo transparency, hindering the tracking of sequential processes like silica-induced macrophage activation, inflammatory factor release, and early fibrotic initiation. Conversely, zebrafish (Danio rerio) presents a promising tool for early toxicity assessment. The transparency of zebrafish embryos allows for clear visualization using immunofluorescence techniques (e.g., labeling neutrophils with GFP or macrophages with mCherry), facilitating real-time tracking of immune cell recruitment and dynamic changes in inflammation and fibrosis [6,11,12]. Additionally, zebrafish exhibit high reproductive capacity (generating 200–300 offspring weekly), substantial genetic homology to humans (sharing 84% of human disease-related genes), and cost-effective maintenance, enhancing their suitability for silicosis research [13,14].

Zebrafish are a valuable model for studying the initial pathological cascades of silica-induced inflammation and fibrosis due to the ability of their hindbrain ventricle (HBV) to mimic the early immune response characteristics of the alveolar microenvironment. Lined with neuroepithelium, the HBV contains a simple epithelial layer, resident macrophages, and quickly recruits peripheral immune cells (neutrophils and monocytes) after exposure to external stimuli, which shared key features with the alveolar microenvironment where silica-induced lung damage initiates [6,15]. This similarity has facilitated the effective modeling of the initial pathological cascades of respiratory diseases such as tuberculosis, COVID-19, as well as silica-induced immune cell–inflammatory–fibrotic initiation in zebrafish [6,14,15,16]. Studies have shown that introducing silica crystals into the zebrafish HBV activates Toll-like receptor (TLR) and Nlrp3 inflammasome signaling pathways, leading to inflammation at the injection site and throughout the body, along with increased expression of pro-fibrotic markers like transforming growth factor-β1 (TGF-β1) and type I collagen [6]. These findings provide a critical theoretical foundation for using zebrafish to dissect the early mechanisms of silicosis toxicity.

Notably, the HBV is a blood–brain barrier-protected central nervous system (CNS) compartment, fundamentally distinct from the open, actively surveillant respiratory epithelium of alveoli. It only mimics the early immune response of alveoli without replicating pulmonary tissue structure, gas exchange, mucosal barrier defense, or interactions with respiratory-specific cell types. Zebrafish serve as valuable models for studying respiratory diseases due to the similarities between their gas bladder and mammalian lungs at both embryological and molecular levels [17,18]. However, the HBV was selected for its transparency and accessibility, enabling real-time monitoring of immune cell recruitment and early inflammatory–fibrotic processes. Moreover, this model has been well-established for inducing early immune activation in response to silica exposure, facilitating comparisons with existing mechanistic data. While the gas bladder model is particularly effective for fibrosis research [17], the HBV model offers greater efficiency in promptly screening initial silica-induced pathology, which aligns with the primary focus of our research.

In this study, respirable α-quartz silica, obtained from the laboratory crushing of natural α-quartz ore, was employed as the test stimulus, with a commercially accessible α-quartz standard utilized as the positive control. Prior to conducting animal experiments, the physicochemical properties of the respirable α-quartz silica were analyzed. Subsequently, the zebrafish embryo model was established by microinjecting the characterized silica into the hindbrain ventricle. The study systematically examined the effects on growth and development, behavioral phenotypes, immune cell recruitment, pathological characteristics, inflammatory factor expression, and fibrotic marker levels. The aim of this research is to assess the potential of respirable α-quartz silica to trigger early immune–inflammatory–fibrotic initiation events in zebrafish embryos, and to evaluate the initial molecular mechanisms of silica toxicity using the HBV model.

2. Materials and Methods

2.1. Fish Husbandry and Embryo Collection

Wild-type zebrafish (AB strain) and transgenic zebrafish lines Tg (mpeg: mCherry) and Tg (mpx: eGFP) were used in this study. The transgenic zebrafish lines expressed mCherry in macrophages (under the mpeg promoter) and eGFP in neutrophils (under the mpx promoter), respectively [19,20]. Healthy 5-month-old zebrafish were maintained in a standardized rearing system with controlled water conductivity (500–1000 μS/cm), temperature (28.0 ± 0.5 °C), and a 14:10 light–dark photoperiod [21]. The fish were fed hatched brine shrimp twice daily. For spawning induction, sexually mature adult fish were placed in spawning chambers at a female-to-male ratio of 1:1 and kept overnight. On the following morning, spawning was initiated via light exposure. Embryos were collected 30 min after stimulation, rinsed thoroughly, and inspected using a stereomicroscope to select morphologically normal individuals. The selected embryos were transferred into 90 mm Petri dishes containing 30 mL of embryo medium (EM: 0.8 g NaCl, 0.04 g KCl, 0.00358 g Na_2_HPO_4_, 0.006 g KH_2_PO_4_, 0.149 g CaCl_2_, 0.246 g MgSO_4_⋅7 H_2_O and 0.35 g NaHCO_3_, and dissolve in 1 L ultrapure water) and incubated until experimentation [21]. All zebrafish care and experimental procedures were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of Guizhou Medical University (Permit number: 2100215).

2.2. Preparation of Silica Crystals

The respirable α-quartz silica test material was made from natural α-quartz ore through a sequential process of crushing, purification, grinding, and size separation. In short, the ore was first crushed stepwise using a jaw crusher (RK/PEF60 × 100, Wuhan Rocker Powder Grinding Equipment Manufacturing Co., Ltd., Wuhan, China), roller crusher (RK/PGS200 × 125, Wuhan Rocker Powder Grinding Equipment Manufacturing Co., Ltd., China), and ceramic grinder (RK/XpM-φ120 mm × 3, Wuhan Rocker Powder Grinding Equipment Manufacturing Co., Ltd., China) to obtain 60–100 mesh particles, followed by water washing and multi-stage magnetic purification (using low- to high-intensity magnetic separators: RK/CRS Φ400 × 300, Wuhan Rocker Powder Grinding Equipment Manufacturing Co., Ltd., China and SLon-100, Ganzhou Jinhuang Magnetic Separation Equipment Co., Ltd., Ganzhou, China). The purified material was dried, ground finely using an agate mortar grinder for 1.5–2.5 h, and the respirable fraction was isolated through repeated hydrostatic sedimentation in deionized water. The final product was obtained after centrifugation and drying at 100–110 °C. For the standard material, α-quartz silica (SRM 1878b) with a median particle size of 3.3 μm was obtained from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA).

2.3. Silica Crystal Injection

Based on a previously established zebrafish model of silicosis, microinjection of 10 mg/mL of crystalline silica into the posterior brain ventricle at 48 hpf has been reported [6,14]. However, our preliminary tests indicated that this concentration of both the standard (SRM 1878b) and test materials resulted in significant zebrafish mortality. Consequently, the exposure concentration was reduced to 5 mg/mL. Respirable α-quartz silica test and standard materials were dissolved in PBS with 0.1% phenol red, followed by sonication for 20 min at 50 W/L and 40 kHz to prepare the final working solutions for subsequent microinjection. Microinjection needles were made from capillary tubes (TW100F-4; outer diameter:1 mm; length: 100 mm), using a micropipette puller (HL-100) from Micrology Precision Instruments Co., Ltd., Wuhan, China. The parameters for pulling were set as follow: Heat, 515, Pull, 80, Velocity, 85, and Pressure, 50. The pulled needles were then sharpened using a micropipette grinder (KDG-03, Micrology Precision Instruments Co., Ltd., Wuhan, China) from the same manufacturer to achieve a tip diameter of approximately 4 μm. For calibration of the injection volume, the microinjection system (DMP-400, Micrology Precision Instruments Co., Ltd., Wuhan, China) from the same manufacturer was utilized, with injection pressure and maintenance pressure set at 18 psi and 0.5 psi, respectively. A droplet of cedarwood oil was placed on a stage micrometer, and a single injection of the sample solution was performed. The resulting sample droplet within the oil measuring approximately 0.125 mm in diameter, confirming an injection volume of 1 nL. Zebrafish embryos at 48 hpf were anesthetized with 0.02% MS-222 and microinjected with 1 nL of sample into the hindbrain ventricle using the microneedle injection parameters described. Control group embryos were injected with PBS containing 0.1% phenol red, while the standard group and test group received 5 mg/mL of the material and the prepared test sample, respectively (n = 3 × 474; 3 replicates, with 474 fish per replicate for each group). After injection, the embryos were transferred into EM supplemented with 1% penicillin-streptomycin (C022, Beyotime, Beijing, China) and maintained until further observation and experimentation [22]. Detailed illustrations of the injection needles and the operational apparatus are available in the Supplementary Materials (Figure S1).

2.4. The Characterization of Silica Particles

X-ray diffraction (XRD) analysis was conducted on a diffractometer using Cu Kα radiation to determine the crystal phase of the SiO_2_ sample. The measurement was performed in the 2θ range from 10° to 80°. Phase identification was achieved by comparing the acquired diffraction pattern with the standard ICDD/JCPDS database.

The surface morphology of the silica particles was characterized by scanning electron microscopy (SEM; S-3400N, Hitachi, Tokyo, Japan). Briefly, the silica exposure suspension was sonicated for 20 min to ensure complete dispersion. An aliquot of 500 µL of the well-dispersed suspension was deposited onto a clean silicon wafer and allowed to dry thoroughly at room temperature. The dried sample was then sputter-coated with a thin layer of gold to enhance conductivity prior to imaging. SEM observations were carried out under high vacuum at an accelerating voltage of 10 kV.

Particle size distribution was measured using a Mastersizer 2000 laser diffraction analyzer (Malvern Panalytical, Malvern, UK). Samples were dispersed in deionized water, sonicated for 5 min, and analyzed with a dispersant refractive index of 1.333.

2.5. Developmental Toxicity Evaluation

Developmental toxicity endpoints, including survival, malformation, and alterations in brain regions, were assessed to characterize the systemic toxicity induced by silica, which corresponds to the clinical extra-pulmonary complications observed in silicosis [23,24]. Following microinjection of respirable α-quartz silica, developmental toxicity was assessed using a stereo microscope (SOPTOP, Ningbo, China). At 72 hpf, survival rate (n = 3 × 96; n = 3 replicates, with 96 fish per replicate for each group), deformity rate (n = 3 × 96) and body length (n = 3 × 15) (Longest linear measurement in rostral-caudal plane) were recorded [25]. At 120 hpf, survival rate was re-evaluated, and morphological analysis included body length measurement, malformation scoring, and examination of forebrain, midbrain, and hindbrain development (n = 3 × 15) [26].

2.6. Behavior Assessment

Behavioral assessments were performed to mirror systemic neurofunctional alterations caused by silica-induced inflammatory cascades, which may reflect the potential extrapulmonary toxic effects triggered by silica exposure beyond local immune responses [23,24]. In this study, three behavioral tests were performed to evaluate the effects of respirable α-quartz silica on zebrafish motor function.

At 72 hpf, zebrafish larvae (excluding those with morphological abnormalities) were individually placed in a 24-well plate (each well containing 2 mL of EM) and acclimated for 15 min. A fine needle was used to gently touch the dorsal side of each larva. The response was observed, including whether the larva exhibited body bending or swimming behavior, and the swimming distance was recorded for responsive larvae. The percentage of responsive larvae and their swimming distance were calculated. The experiment was repeated three times, with 15 larvae per treatment group in each replicate [27].

Locomotor activity was assessed in healthy 120 hpf zebrafish larvae, which were individually transferred to a 24-well plate containing 2 mL of embryo medium (EM) per well. Behavioral tracking was conducted using a ZebraLab system (version 3.5, ViewPoint Life Science, Lissieu, France) under a light–dark stimulation protocol established in our previous study [28]. After a 10-min acclimation period under light conditions, larvae were subjected to a 20-min testing cycle consisting of alternating 5-min light and 5-min dark periods. The tracking module was configured to acquire data at 1-min intervals with light intensity maintained at 100% during light phases. Swimming distance and speed were recorded throughout the 20-min cycle, with data from the initial 10-min acclimation period excluded from analysis. Velocity and distance measurements were calculated for each 1-min interval and averaged separately for light and dark phases (n = 3 × 24).

Anxiety-like behavior in healthy 120 hpf zebrafish larvae was evaluated using the same ZebraLab system [29]. Larvae were individually transferred to a 24-well plate with each well containing 2 mL of EM. After a 20-min acclimation period in darkness, locomotor activity was recorded during a subsequent 10-min dark phase. Using the ZebraLab software (version 3.5), each 16 mm diameter well was virtually divided into an inner zone (8 mm diameter) and an outer zone. The number of crossings between zones per minute per larva and the percentage of time spent in the inner zone during the 10-min dark period were calculated to quantify anxiety-like behavior (n = 3 × 24).

2.7. In Vivo Imaging of Inflammatory Cells

To investigate the recruitment of macrophages and neutrophils to the injection site, 1 nL of respirable α-quartz silica was microinjected into the hindbrain ventricle of Tg (mpeg:mCherry) and Tg (mpx:eGFP) transgenic zebrafish at 48 hpf. Fluorescence images were captured at 6, 12, 18, 24, 36, and 72 h after microinjection using a Nikon Ts2R fluorescence microscope (Nikon, Shinagawa-ku, Japan) with excitation/emission wavelengths set at 587/610 nm for mpeg (macrophages) and 455/505 nm for mpx (neutrophils). Fluorescence intensity in the hindbrain ventricle was quantified using Image J 2.14.0, and relative fluorescence intensity was calculated by normalizing against the control group (n = 3 × 6).

2.8. Histological Analysis

Histological examinations, including H&E (n = 3 × 6, three biological replicates, 6 larvae per replicate, with at least 3 technical sections per fish, total 18 larvae or 54 sections per group), Masson’s trichrome (n = 3 × 6), and Sirius red staining (n = 3 × 6), were conducted to evaluate fibrosis and collagen deposition in the zebrafish brain. Healthy zebrafish larvae at different developmental stages (72 and 120 hpf) were fixed in 4% paraformaldehyde for 48 h. Subsequently, the larvae were dehydrated through a graded ethanol series, cleared with xylene, and embedded in molten paraffin. Serial sections of 4 μm thickness were cut using a HistoCore Multicut microtome (Leica Microsystems, Wetzlar, Germany). Prior to histological staining, the sections were deparaffinized in xylene, rehydrated through a descending ethanol series to distilled water, and then processed for subsequent pathological examinations. For H&E staining (G1120, Solarbio, Beijing, China), tissue sections were stained with hematoxylin for 1 min, differentiated in 0.1% acid alcohol, rinsed in distilled water, counterstained with eosin for 30 s, dehydrated through a graded ethanol series, cleared in xylene, and mounted with neutral balsam.

For Masson’s trichrome staining, deparaffinized sections were stained following a standard procedure (G1340, Solarbio, China). Nuclei were stained with Weigert’s hematoxylin for 5 min and blued for 3 min. Subsequently, sections were stained with Ponceau-Acid Fuchsin for 7 min, treated with phosphomolybdie acid for 1 min for differentiation, and then counterstained with Aniline Blue for 1 min to visualize collagen. After each step, sections were rinsed appropriately. Finally, sections were dehydrated, cleared in xylene, and mounted with neutral balsam.

For Sirius red staining (G1472, Solarbio, China), deparaffinized sections were sequentially stained with iron hematoxylin for 10 min and Sirius Red solution for 15 min. After a quick rinse in distilled water, sections were dehydrated through a graded ethanol series, cleared in xylene, and mounted with neutral balsam.

All stained sections were digitally scanned using a NanoZoomer S60 digital slide scanner (Hamamatsu, Japan) for the evaluation of fibrosis and collagen deposition in the zebrafish brain. Image quantification was conducted using the open-source software NDP.view 2 (Hamamatsu, Japan). Download link: https://www.hamamatsu.com.cn/cn/zh-cn/product/life-science-and-medical-systems/digital-slide-scanner/U12388-01.html (accessed on 28 July 2025).

2.9. Hydroxyproline Assay

The collagen content in healthy zebrafish brain tissue was assessed using a hydroxyproline assay kit (A030-2-1, Nanjing, China), following the manufacturer’s instructions. Briefly, the head tissue of larvae (n = 3 × 150) at 120 hpf was homogenized in lysis buffer and heated in a boiling water bath for 20 min. Then, 0.5 mL of Reagent 1 was added, mixed, and allowed to stand for 10 min. Subsequently, 0.5 mL of Reagent 2 was added, mixed, and incubated for 5 min. Next, 0.5 mL of Reagent 3 was added, and the mixture was incubated at 60 °C for 15 min. After cooling, the sample was centrifuged at 3500 rpm for 10 min. The supernatant was collected, and the absorbance was measured at a wavelength of 550 nm to calculate the collagen content.

2.10. RNA Extraction and Quantitative Real-Time PCR Analysis

Total RNA was extracted from pooled brain tissues of healthy zebrafish at 120 hpf (150 larvae per replicate) using TRIzol reagent (Life Technologies, Waltham, MA, USA). After quality verification (A260/A280: 1.8–2.0), 3000 ng of RNA was reverse-transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) in a 10 µL reaction. qPCR was performed in 10 µL volumes containing TB Green Premix Ex Taq II (Takara, Shiga, Japan), 0.8 µL cDNA, and gene-specific primers (10 µM) on a CFX96 system (Bio-Rad, Hercules, CA, USA). The thermal cycling protocol was performed as previously described [30]. Gene expression was analyzed via the 2^−ΔΔCT^ method normalized to β-actin, with all primers (Supplementary Table S1) synthesized by Shenggong Biotech (Shanghai, China).

2.11. Statistical Analysis

All data are expressed as mean ± SEM. Statistical analyses were performed under double-blind conditions using SPSS software (version 23.0), while graphs were generated with GraphPad Prism (version 9.0). To ensure blinding, personnel performing respirable α-quartz silica microinjection were separated from those conducting endpoint measurements, and all participants remained unaware of the group assignments. Normality of data distribution was assessed using the Kolmogorov–Smirnov test (normality criterion: p > 0.10), and homogeneity of variances was evaluated using Levene’s test (homoscedasticity criterion: p > 0.05). Multiple group comparisons were carried out using one-way analysis of variance (ANOVA). If significant differences were detected (p < 0.05), post hoc pairwise comparisons were conducted using the Bonferroni correction.

3. Results

3.1. Structural and Compositional Analysis of Respirable α-Quartz Silica

The test sample was successfully obtained from natural α-quartz ore via a process of crushing, purification, grinding, hydro-sedimentation, and centrifugal separation. XRD analysis confirmed that the sample was predominantly composed of SiO_2_ (98.62%). The XRD pattern exhibited sharp and well-defined diffraction peaks, which are characteristic of highly crystalline α-quartz. Chemical analysis identified minor impurities, including Al_2_O_3_, Fe_2_O_3_, CaO, and MgO. Particle size analysis showed a predominant distribution in the range of 2.78 to 4.12 μm (Figure 1A). SEM analysis revealed that the reference standard exhibited a granular morphology with well-defined boundaries, while the test sample presented with indistinct particle edges (Figure 1B). Laser diffraction measurements yielded particle sizes of 3.217 μm for the standard and 3.056 μm for the test sample (Figure 1C,D).

3.2. Developmental Toxicity

Following microinjection of respirable α-quartz silica, zebrafish in both the standard and test groups exhibited significantly decreased survival rates (p = 0.021 for standard and p = 0.039 for test at 72 hpf; p = 0.014 for standard and p = 0.016 for test at 120 hpf) and significantly increased deformity rates (p = 0.014 for standard and p = 0.024 for test at 72 hpf; p = 0.001 for standard and p = 0.008 for test at 120 hpf) (Figure 2A,B). However, no significant differences in body length were observed among the treatment groups compared to the control group at 72, 96, and 120 hpf (Figure 2D). Analysis of the brain at 120 hpf revealed that the treatment groups showed a significant increase in midbrain (p = 0.016 for standard; p = 0.027 for test) and hindbrain (p = 0.018 for standard; p = 0.038 for test) lengths compared to the control group (Figure 2E–G).

3.3. Locomotor Behaviors of Larval Zebrafish

Behavioral assessments revealed significant neurotoxic effects. At 72 hpf, the touch response rate (p = 0.034 for standard; p = 0.049 for test) was significantly decreased in all treatment groups (Figure 3B). However, only the standard group exhibited a significant reduction in the swimming distance post-stimulation (p = 0.014) compared to the control (Figure 3C). In the light–dark transition test at 120 hpf, the average movement velocity of all treatment groups was significantly decreased solely during the first light period (p = 0.021 for standard; p = 0.026 for test), with no significant differences observed in subsequent cycles (Figure 3E,F). Furthermore, in the open field test at 120 hpf, all treatment groups spent significantly less time in the central zone than the control group (p = 0.018 for standard; p = 0.024 for test) (Figure 3I).

3.4. Recruitment of Immune Cells

The inflammatory response initiated by SiO_2_ microinjection exhibited distinct phases. A wave of neutrophil recruitment was observed in Tg (mpx: eGFP) zebrafish, beginning as early as 6 h after injection, peaking at 18 h, and then subsiding (Figure 4B). This was followed by a macrophage response in Tg (mpeg: mCherry) zebrafish, which commenced at 12 h after injection and crested at 24 h (Figure 4C).

3.5. Histopathological Analysis of the Zebrafish Brain

Histopathological analysis indicated the absence of discernible collagen deposition in H&E-stained, Masson’s trichrome and Sirius red-stained sections, and we did not observe any fibrosis in zebrafish brain (Figure 5).

3.6. Measurement of Inflammatory and Fibrotic Factors

qPCR analysis revealed a significant upregulation of key inflammatory [tnf-α (p = 0.029 for standard; p = 0.002 for test), il-6 (p = 0.017 for standard; p = 0.022 for test), il-1β (p = 0.024 for test)] and fibrosis-related genes (tgf-β (p = 0.001 for standard; p < 0.001 for test), acta-2 (p = 0.022 for standard; p = 0.002 for test), collagen (p = 0.026 for standard; p = 0.001 for test)) in the brain tissue of all treatment groups compared to the control (Figure 6A). Consistent with the genetic profile, the biochemical assessment confirmed an increase in hydroxyproline content, a clinical gold standard for fibrosis, within the brain tissue (p = 0.019 for standard; p = 0.009 for test) (Figure 6B).

4. Discussion

This study systematically investigated the effects of respirable α-quartz silica on zebrafish, and the findings confirmed its ability to induce pathological and pathophysiological changes associated with the early initiation of silicosis in zebrafish, including particle characteristics, growth index, behavioral alterations, tissue morphology, and molecular responses.

This study synthesized and characterized the properties of α-quartz particles, comparing respirable α-quartz silica particles with an average size of 3.056 μm to the standard size of 3.217 μm. The size of these particles is critical for their potential pulmonary toxicity, as particles smaller than 10 μm are thought to bypass the respiratory mucociliary defense system and accumulate in the alveolar region [31], as mimicked by the hindbrain ventricle in the zebrafish model employed in this study. Particles with a diameter of approximately 3 μm are susceptible to macrophage phagocytosis, leading to subsequent pathological processes. Despite minor size discrepancies, these particles, meeting pathogenic criteria, provide a consistent stimulus for assessing their effects on zebrafish.

Injection of respirable α-quartz silica into the 2 dpf zebrafish hindbrain ventricle did not affect body length but led to abnormal midbrain and afterbrain width changes in zebrafish at 96 hpf and 120 hpf. Elevated mortality and malformation rates suggest that α-quartz silica disrupts zebrafish embryonic development, potentially causing systemic developmental toxicity beyond local organ effects. The disturbances in embryonic development may be attributed to two main factors: firstly, the particles induce an inflammatory response that impedes cell proliferation and differentiation by depleting energy and nutrients, as supported by the subsequent molecular-level investigations; secondly, the particles can spread systemically through the bloodstream, leading to direct harm to multiple organ primordia and resulting in developmental delays. These findings align with studies in mammalian models indicating that exposure to silica results in abnormal embryonic development [32,33], highlighting the broad toxicity of α-quartz silica and providing a model-based framework for understanding the systemic complications in individuals with silicosis, such as increased risk of tuberculosis [34,35], nephritis and scleroderma [24]. It should be noted that these data offer additional evidence for the subsequent main findings regarding immune cell recruitment and the initiation of inflammatory–fibrotic processes, rather than being specific to silicosis phenotypes.

Behavioral results revealed that α-quartz free silica decreased the total distance moved in light, reduced the touch response, and increased activity mainly in the periphery of the six-well plate. The aberrant behaviors observed may be associated with the presence of α-quartz in the hindbrain, a critical region for zebrafish neural development responsible for motor coordination and stress response regulation [36,37]. Furthermore, local inflammation or tissue damage could also contribute to these effects. It should be emphasized that these phenotypes are specific to the model and influenced by local inflammation, rather than accurately reflecting the neurofunctional complications seen in human silicosis. The resemblance in behavioral patterns between the positive control group and the α-quartz free silica group indicates that the alterations in behavior are probably caused by α-quartz itself, rather than other factors, establishing a behavioral phenotype foundation for subsequent investigation into early pathological mechanisms of silica-induced damage in this model. Notably, the neurobehavioral alterations are mechanistically associated with subsequent silica-induced systemic inflammation (elevated il-6, tnf-α, and il-1β), a pathway crucial for both neurofunctional disruptions and silicosis pathogenesis, providing contextual backing for core inflammatory–fibrotic results rather than serving as direct indicators of pulmonary conditions.

Despite the absence of organs like lungs, zebrafish display a unique anatomical trait in the form of a fluid-filled hindbrain ventricle, which has the ability to recruit immune cells and mimic the early immune response characteristics of the alveolar microenvironment. When exposed to α-quartz silica, zebrafish embryos develop abnormalities that closely resemble the early stages of silicosis in humans, evidenced by the presence of particles in alveoli and the infiltration of inflammatory cells [6,14]. In this study, neutrophils rapidly gather near the hindbrain ventricle region shortly after injection, initiating an acute inflammatory response that peaks at 18 h post-injection and gradually diminishes thereafter. Conversely, macrophages exhibit delayed recruitment towards the hindbrain ventricle, reaching peak levels 24 h post-injection before gradually declining. These activated macrophages, crucial for engulfing silica particles, release various pro-inflammatory and pro-fibrotic factors. However, the transition from inflammation to fibrosis in the hindbrain ventricle of zebrafish following prolonged stimulation remains poorly understood.

α-quartz particles upregulate inflammatory cytokines like il-6, tnf-α, and il-1β expression. Persistent inflammation can activate signaling pathways leading to increased tgf-β expression, resulting in myofibroblast activation, as indicated by acta-2 expression, and collagen accumulation. Elevated levels of fibrotic markers confirm the capacity of respirable α-quartz silica to initiate an inflammation–fibrosis cascade in the hindbrain ventricle region of zebrafish, mimicking the early pathogenesis of silicosis in humans [6]. Furthermore, our findings indicate that respirable α-quartz silica induces more pronounced inflammation and fibrosis-related markers compared to the positive control group, suggesting a heightened fibrotic risk potentially linked to the smaller particle size of α-quartz free silica in comparison to the positive control material. In addition, ELISA analysis showed elevated hydroxyproline levels, a crucial component in collagen synthesis, indicating increased collagen metabolism triggered by inflammatory mediators. This observation supports the activation of fibrotic pathways associated with collagen metabolism by α-quartz free silica. Despite changes in inflammatory and fibrotic markers, as well as hydroxyproline levels, no notable morphological abnormalities were observed in the zebrafish hindbrain ventricle region. Moreover, no significant changes in tissue morphology or fibrotic structure were observed in the surrounding tissues of the hindbrain ventricle region, confirming that this model captures early-onset fibrosis-related events rather than mature fibrosis. Although paraffin-embedded staining effectively identified initial collagen deposition in this investigation, utilizing resin-embedded semithin sections and transmission electron microscopy (TEM) may offer enhanced resolution for subcellular pathology examination.

This study has several limitations. Firstly, zebrafish lack true lung organs. While the hindbrain ventricle can mimic certain aspects of the alveolar environment, it cannot fully replicate the intricate structure of human lung tissue, especially the alveolar–capillary barrier. Secondly, the short lifespan of the zebrafish hindbrain ventricle limits this study to capturing the initial silicosis changes, such as inflammation onset and a fibrosis-like phenotype, without modeling the chronic progression observed in human silicosis over decades. The observation window of this study (up to 120 hpf) only captures the early inflammatory–fibrotic initiation phase of silica-induced injury, which is fundamentally distinct from the decades-long pathological progression of clinical silicosis. Thirdly, the study did not utilize the zebrafish gas bladder, a tissue with established lung homology suitable for modeling respiratory disease; therefore, future studies should compare results between HBV and gas bladder models to validate translational relevance. Fourthly, direct bolus microinjection of silica into the HBV is a simplified experimental approach that does not mimic the physiological process of silica particle translocation to the brain in human silicosis, where particles are inhaled through respiration and can disseminate systemically over long-term exposure. In addition, neurobehavioral changes seen in this model result from local inflammation with the HBV, with no direct evidence linking them to human silicosis-related neurofunctional impairments. It is advisable to refrain from overinterpreting these behavioral data as a model for silicosis-related neurodegeneration. Lastly, the study primarily examines early molecular and phenotypic changes, necessitating further validation in mammalian models to confirm translational relevance in understanding human silica-induced lung injury.

5. Conclusions

In summary, this study provides mechanistic insights into the early immune cell–inflammatory–fibrotic initiation induced by respirable α-quartz silica (3.056 μm) in zebrafish embryos. Key findings include sequential recruitment of neutrophils and macrophages, upregulation of core inflammatory (tnf-α, il-6, il-1β) and fibrotic (tgf-β, acta-2, collagen) genes, and early pathophysiological changes (developmental toxicity, behavioral alterations, elevated hydroxyproline) within 5 days. The 120-h observation period was selected according to the temporal response profile in our model, encompassing the peak of immune cell recruitment and the subsequent activation of inflammatory–fibrotic signaling cascades. This timeframe is optimal for capturing the critical early events of silica-induced toxicity, directly supporting the value of this model for identifying the early silica-triggered signaling pathway. Notably, this work is not a silicosis disease model nor does it directly inform occupational health assessment. Its core value lies in identifying early silica-induced signaling pathways for future silica toxicity research. Future studies should validate these pathways in mammalian chronic exposure models, employ zebrafish gas bladder models to mimic pulmonary-specific responses, and integrate occupational population data to link early molecular events with long-term outcomes, enabling comprehensive elucidation of silicosis mechanisms and meaningful occupational health protection.