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. Author manuscript; available in PMC: 2025 May 3.
Published in final edited form as: Exp Eye Res. 2023 Jul 13;234:109575. doi: 10.1016/j.exer.2023.109575

Toxicological effects of ocular acrolein exposure to eyelids in rabbits in vivo

Suneel Gupta a,b, Lynn M Martin a,b, Eric Zhang a,c, Prashant R Sinha a,b, James Landreneau a,c, Nishant R Sinha a,b, Nathan P Hesemann a,c, Rajiv R Mohan a,b,c,*
PMCID: PMC12049013  NIHMSID: NIHMS2061973  PMID: 37451567

Abstract

Acrolein is a highly reactive volatile toxic chemical that injures the eyes and many organs. It has been used in wars and terrorism for wounding masses on multiple occasions and is readily accessible commercially. Our earlier studies revealed acrolein’s toxicity to the cornea and witnessed damage to other ocular tissues. Eyelids play a vital role in keeping eyes mobile, moist, lubricated, and functional utilizing a range of diverse lipids produced by the Meibomian glands located in the upper and lower eyelids. This study sought to investigate acrolein’s toxicity to eyelid tissues by studying the expression of inflammatory and lipid markers in rabbit eyes in vivo utilizing our reported vapor-cap model. The study was approved by the institutional animal care and use committees and followed ARVO guidelines. Twelve New Zealand White Rabbits were divided into 3 groups: Naïve (group 1), 1-min acrolein exposure (group 2), or 3-min acrolein exposure (group 3). The toxicological effects of acrolein on ocular health in live animals were monitored with regular clinical eye exams and intraocular pressure measurements and eyelid tissues post-euthanasia were subjected to H&E and Masson’s trichrome histology and qRT-PCR analysis. Clinical eye examinations witnessed severely swollen eyelids, abnormal ocular discharge, chemosis, and elevated intraocular pressure (p < 0.001) in acrolein-exposed eyes. Histological studies supported clinical findings and exhibited noticeable changes in eyelid tissue morphology. Gene expression studies exhibited significantly increased expression of inflammatory and lipid mediators (LOX, PAF, Cox-2, and LTB4; p < 0.001) in acrolein-exposed eyelid tissues compared to naïve eyelid tissues. The results suggest that acrolein exposure to the eyes causes acute damage to eyelids by altering inflammatory and lipid mediators in vivo.

Keywords: Acrolein, Eyelids, Lipids, Vapor cap method, Inflammation

1. Introduction

Acrolein, an unsaturated volatile aldehyde, is a strong irritant and is known to cause severe problems in the eye, skin, and lungs. It is used as an intermediate compound in various industries for the production of different chemicals (Gupta et al., 2021). Acrolein is a disruptive by-product and a ubiquitous pollutant in the environment from the combustion of plastic products, cigarette smoke, and overheating of oils. In a cumulative production amount, the United States produces about 500 million pounds of acrolein annually (Ashizawa et al., 2007). Acrolein was used as a warfare agent (“papite’) in World War I (Ghilarducci and Tjeerdema, 1995). The Department of Homeland Security, Agency for Toxic Substances and Disease Registry, and Environmental Protection Agency have listed acrolein as a high-priority toxic chemical (Toxicological Profile For Acrolein, 2007; Moghe et al., 2015). Acrolein’s toxicity to the skin, lungs, and heart are well studied (Ashizawa et al., 2007; Dachir et al., 2015; Feng et al., 2006; Gupta et al., 2020; Jia et al., 2009; Moghe et al., 2015) but limited information about its toxicity to eyes especially for eyelids is available currently.

The eyelid contains a tarsal plate with dense fibrous connective tissue, meibomian glands, follicles, muscular bundles, and the surface epithelium. The eyelid plays an important role in maintaining tear film and ocular surface homeostasis (Dartt and Willcox, 2013; Sun et al., 2018). It regulates lipid biosynthesis and secretes substances to keep the ocular surface moist and lubricated through lipids secreted by the meibomian glands (Robin et al., 1985; Wolff, 1946, 1954). In lipid biosynthesis, lipid mediators are important endogenous regulators of neural cell proliferation, differentiation, oxidative stress, inflammation, and apoptosis (Farooqui, 2009). Lipids and lipid metabolism play an important role in maintaining the function of the eye. The eyelid is the first affected tissue after ocular chemical injury which leads to a variety of ocular disorders (Amescua et al., 2019; Carter, 1998; Chisholm et al., 2017; Sabeti et al., 2020).

Acrolein exposure to the eye is known to cause severe ocular discomfort, corneal morbidity, corneal inflammation, corneal haze, corneal neovascularization, and vision impairment (Dachir et al., 2015; Gupta et al., 2020; McCracken et al., 2014). The cellular toxicity to tissue from acrolein is linked to the change in reactive oxygen species (ROS), glutathione, and wound healing patterns. Under cellular trauma, a variety of compounds are formed during ROS and lipid peroxidation (LPO) of polyunsaturated fatty acids of membrane phospholipids. Acquiring literature indicates that strong electrophilic properties of acrolein and its high reactivity with nucleophiles in all α-, and β-unsaturated aldehydes (Esterbauer et al., 1991; Uchida et al., 1998), can alter the expression of inflammatory markers, lipids, lipid mediators, and lipid biosynthesis. Acrolein toxicity is mainly due to the alterations in several cell functions, which mostly depend on the formation of covalent adducts with cellular proteins (Pizzimenti et al., 2013; Kashiwagi and Igarashi, 2023). Acrolein and its adducts have been linked to different pathological conditions like diabetic nephropathy; renal disease; Sjogren’s syndrome; cerebral stroke/infarction; colon cancer; and diabetes (Moghe et al., 2015). The literature reveals that acrolein in non-ocular tissues mediates ROS and LPO production and triggers the overactivation of immune cells and inflammation and these phenomenon increases the production of lipid mediators that act through specific receptors (Wójcik et al., 2021). The research shows that during oxidants assault lipids and polyunsaturated fatty acids generate toxic lipid aldehydes and alter the cellular functioning that leads to neurological disorders (Taso et al., 2019).

The current study investigated acrolein’s toxicity to eyelids by studying the expression of a few selected inflammatory and lipid markers in vivo in rabbit eyes exposed to acrolein vapor for two different conditions employing an optimized vapor-cap technique for rabbit eyes. Our central hypothesis is that an increase in lipid peroxidase in eyelid tissue post acrolein exposure compromises the normal expression and functioning of inflammatory, lipids, and lipid modulators in the eye.

2. Materials and methods

2.1. Chemicals and reagents

Acrolein (RCC150) was purchased from ULTRA Scientific Inc. (Thermo Fisher, Grand Island, NY, USA) to evaluate its toxicity to eyelid tissue. Topical artificial tears were purchased from the Rugby Laboratories (Livonia, MI, USA), and sterile Weck-Cel ophthalmic spears from Beaver-Visitec International Inc. (Waltham, MA). Ketamine hydrochloride (JHP Pharmaceuticals, LLC, Rochester, MI, USA), xylazine hydrochloride (XylaMed, Bimeda Inc., IL, USA), 0.5% proparacaine hydrochloride drops (Alcon, Ft. Worth, TX, USA) and euthasol solution (051311–050-01, Virbac, Penn Veterinary Supply, Inc. Lancaster, PA, USA) were obtained from the pharmacy of the Harry S. Truman Memorial Veterans’ Hospital, Columbia, Missouri, USA. Hematoxylin and eosin (H&E) and Masson’s trichrome staining solutions were procured from StatLab (McKinney, TX, USA), balanced salt solution (BSS) and 2-Methyl butane from Thermo Fisher (Grand Island, NY, USA), CD11b (cat # BDB550282) from BD Pharmingen (San Jose, CA), and commercial toxicity assay kits from different vendors.

2.2. Animals

The Institutional Animal Care and Use Committees of the University of Missouri and the Harry S. Truman Memorial Veterans’ Hospital approved the study. Animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Twelve New Zealand White Rabbits (4–5 pounds; Charles River Laboratory Inc., Wilmington, MA) were divided into 3 groups: Naïve (group 1, n = 4), 1-min acrolein exposure (group 2, n = 4), or 3-min acrolein exposure (group 3, n = 4). Rabbits were housed in temperature-controlled (21 ± 1 °C) rooms with a light-dark cycle for 12 h and had ad libitum access to food and water. Only female rabbits were used as gender does not significantly impact corneal wound healing events in rabbits (Tripathi et al., 2019). Rabbits were anesthetized with an intraperitoneal injection of ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (10 mg/kg) cocktail and received 2 drops of topical anesthetic, proparacaine hydrochloride (0.5%), on the eye before any procedure to minimize pain and discomfort. Only one eye of each animal was used for studying acrolein toxicity to the eyes. Rabbits were thermally supported throughout the procedure and during the anesthetic recovery period.

2.3. Acrolein exposure procedure

Rabbit eyes were exposed to acrolein vapors for 1-min or 3-min following using a vapor-cap exposure protocol (Gupta et al., 2020). In brief, an 8-mm diameter filter paper disc containing acrolein (30 μL) was placed in the center of a circular clear polypropylene cup and then positioned upside-down onto the rabbit eye (Supplementary Fig. 1). This setup allowed ocular surface exposure to acrolein’s vapors. Right after exposure, the eye was profusely washed with 20 ml of BSS once.

2.4. Clinical eye examinations and imaging

The clinical eye examinations and imaging were performed in live animals before and after acrolein exposure at 30-min, day-1, day-3, day-7, and day-14 under general anesthesia as reported previously (Gupta et al., 2017). A stereomicroscope (Leica MZ16F, Leica Microsystems Inc., Buffalo Grove, IL, USA) equipped with a digital camera (SpotCam RT KE, Diagnostic Instruments Inc., Sterling Heights, MI, USA) was used to assess changes in eyelids pre/post acrolein. All clinical eye exams were performed by a minimum of two independent investigators (SG, LMM, EZ, or NPH) in a masked manner. Eyes were kept moist during the procedure with BSS. A handheld tonometer (Tono-Pen AVIA® Tonometer) was used to record intraocular pressure (IOP) before and after acrolein exposure at selected times following the reported method (Gupta et al., 2022; Sharma et al., 2016). Schirmer Tear Test Strip (Fisher Scientific, Grand Island, NY, USA) was used to quantify tear volume at selected time points as reported previously (Gupta et al., 2022).

2.5. Eyelid tissue collection

Rabbits were humanely euthanized by an intravenous injection of euthasol solution (150 mg/kg) while animals were under general anesthesia. Upper and lower eyelid tissues were collected under a dissecting microscope (Leica Wild M690, Leica Microsystems Inc., Buffalo Grove, IL, USA) as reported earlier (Asano et al., 2017). Tissues were immediately placed into molds containing optimal cutting temperature (OCT) compound or in RLT buffer, snap-frozen, and maintained at −80 °C until further processing. Eight-micron-thick sections were prepared with a cryo-microtome, mounted on glass slides, and used for analysis. Eyelid tissues stored in RLT buffer were used to isolate RNA and generate cDNA for gene expression studies.

2.6. Histopathological and toxicological assays

2.6.1. H&E and Masson’s trichrome staining

Rabbit eyelid sections from the naïve and acrolein-exposed groups were used in H&E staining and Masson’s trichrome stainings to study the effects of acrolein on cellular morphology and collagens in eyelids, respectively, following the reported method in our laboratory (Balne et al., 2021; Gupta et al., 2018; Zhang et al., 2022). Additionally, the Veterinary Medical Diagnostic Laboratory at the University of Missouri was also used for these histological analyses for higher rigor.

2.6.2. CD11b immunofluorescence

To study the toxicity of acrolein on the eyelid tissue CD11b immunofluorescence was performed following the reported method (Gupta et al., 2022). Eyelid tissue sections were incubated with the primary CD11b antibody (BDB550282) at 1:100 dilution in a 1X HEPES buffer containing 2% BSA for 90 min, followed by goat anti-rat IgG secondary antibody Alexa Fluor 594 (A11007; Invitrogen) at 1:500 dilution for 60 min at room temperature. Thereafter, sections were mounted in an antifade medium with DAPI (H1200; Vector lab), viewed, and photographed under a fluorescence microscope (Leica, Deerfield, IL) equipped with a digital camera system (SpotCam RT KE; Diagnostic Instruments, Sterling, MI).

2.6.3. Reactive oxygen species (ROS) assay

The levels of intracellular reactive oxygen species (ROS) in the eyelid tissue were examined using the OxiSelect™ Intracellular ROS assay kit (STA-342; Cell Biolabs, Inc, CA, USA) following the reported method (Gupta et al., 2021). In brief, rabbit eyelid tissue sections were kept at room temperature followed by 3 washing (5 min each) with phosphate-buffered saline solution (PBS), and incubation in 2′, 7′-dichloro-dihydro fluorescin diacetates (1X; DCFH-DA) solution for 15 min at room temperature. Thereafter, the tissue sections were mounted, viewed, and photographed under a fluorescence microscope (Leica) equipped with an imaging system (SpotCam).

2.6.4. Lipid peroxidation (LPO) assay

To study the effects of acrolein on free radical-mediated injury to eyelids, lipid peroxidation (LPO) measurements were performed using a commercial LPO kit (705002; Cayman Chemicals, Inc, MI, USA) following the manufacturer’s guidelines. The tissue lysates were prepared according to the vendor’s instructions. The hydroperoxide concentration in samples was calculated against the lipid hydroperoxide standard.

2.6.5. Leukotriene B4 (LTB4) assay

The LTB4 is considered a potent mediator of inflammation and activates leukocyte functions, superoxide anion production, and chemotaxis. Thus, the impact of acrolein exposure on the modulation of these parameters in eyelid tissues was studied using LTB4 Express ELISA Kit (10009292; Cayman Chemicals, Inc, MI, USA). The tissue lysates and assay were performed following the manufacturer’s guidelines. The LTB4 activity was determined by measuring and absorbance at 405 nm using an Epoch BioTek plate reader (BioTek Instruments, VT, USA). The LTB4 concentration in samples was calculated against the LTB4 standard.

2.7. Quantitative qRT-PCR analysis

The effects of acrolein on COX2, LOX, and PAF expressions were evaluated at the mRNA level by running quantitative reverse transcription PCR reactions in the QuantStudio 6 Flex™ Real-Time PCR System (Applied Biosystems, Carlsbad, CA). The mRNA was extracted using the RNeasy Mini kit (Qiagen, Valencia, CA) as reported previously (Anumanthan et al., 2018; Chaudhary et al., 2014), and cDNA was prepared using 2 μg of mRNA using a reverse transcription system (Promega, Madison, WI) as reported earlier (Gupta et al., 2018). A 20 μL qPCR reaction mixture contained 2 μL of cDNA, 2 μL of 200 nM forward primer, 2 μL of 200 nM reverse primer, 10 μL of 2X SYBR green supermix (Bio-Rad Laboratories, Hercules, CA), and 4 μL of RNAse/DNAse free water. The qRT-PCR was run at universal cycle conditions, including initial denaturation at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s, annealing, and extension at 60 °C for 60 s. The beta-actin (β-actin) was used as the housekeeping gene and data were normalized with naïve control. The relative mRNA expression was calculated using the 2—ΔΔCt method and reported as a relative fold change to the corresponding control values.

2.8. Statistical analysis

GraphPad Prism 9.2 software (GraphPad, La Jolla, CA USA) software was used for the statistical analysis. The Student’s t-test and one-way analysis of variance (ANOVA) with the Wilcoxon rank sum test, or the Bonferroni multiple comparison posthoc test were used depending on the experimental design for quantification data collected for IOP, tear flow, and different bioassays to determine statistical power. The p values < 0.05 or lower were considered statistically significant.

3. Results

3.1. In-situ biomicroscopy evaluation

The stereo-biomicroscopy evaluation showed severe ocular inflammation, lacrimation, redness, and swelling in eyelids after acrolein exposure whereas vehicle-exposed and naive eyes showed no pathologies (Fig. 1). The toxicological effects of ocular acrolein were dose-and time-dependent. Eyes exposed for 3 min of acrolein exhibited greater morbidity to eyelids compared to 1 min (Fig. 1B–E and Fig. 1G–J), particularly on day-1, and day-3 compared to day-7 and day-14 (Fig. 1B–E and Fig. 1G–J). Further, acrolein-exposed eyes showed blepharitis, conjunctival neovascularization, and secretions on the eyelid margins.

Fig. 1.

Fig. 1.

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In vivo stereo-biomicroscopy images show severe eyelid inflammation in the rabbit eyes following acrolein vapor exposure for 1 min (B–E) and 3 min (G–J) up to 14 days represented by the black arrow sign. Images A and F represent the control non-exposed (baseline) before exposure to rabbit eyes. n = 4 rabbit eyes in each group. All images were taken at 7.1x magnification. Scale bar = 3.0 mm.

3.2. Clinical evaluation

The IOP and tear volume were recorded during the clinical eye examinations. Acrolein-exposed eyes showed significantly increased IOP at all tested time points compared to vehicle-exposed and naive eyes (Fig. 2). An increased tearing was observed in acrolein-exposed eyes until day-7 compared to vehicle-exposed and naive eyes (Fig. 3). Tearing was greater in eyes exposed to acrolein for 3-min than 1-min. Interestingly, the tear volume increased significantly up to day-3 in both acrolein exposure duration and reduced later in day-10, and day-14 time points (Fig. 3A, and B).

Fig. 2.

Fig. 2.

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Line graphs reveal that acrolein exposures (1-, and 3-min.) significantly increased intraocular pressure (IOP) in rabbit eyes (A and B) compared to the naïve control (baseline) group. The results were presented as mean ± SD. n = 4 rabbit eyes in each group. The Student’s t-test was used for statistical analysis. ***p ≤ 0.001.

Fig. 3.

Fig. 3.

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Line graphs depict that acrolein exposures (1-, and 3-min.) amend the tear flow in rabbit eyes compared to the naïve control (baseline) group (A and B). During the initial three examination time points, (4 h, day 1, and day 3) significantly increased tear flow was recorded as regular irritation symptoms of acrolein exposure. Contrary, the tear flow decreased significantly in later time points (day-10, and day 14). The results were presented as mean ± SD. n = 4 rabbit eyes in each group. The Student’s t-tests test was used for statistical analysis. *p ≤ 0.05; **p ≤ 0.01; and ***p ≤ 0.001.

3.3. Histopathological staining and immunofluorescence evaluations

The H&E staining of the eyelid tissues showed notable damage to epithelial cells, orbicular muscles, and meibomian glands in acrolein-exposed eyes compared to vehicle-exposed and naive eyes (Fig. 4). Likewise, the Masson’s Trichrome staining revealed of appreciably higher blue staining in acrolein exposed eyes compared to vehicle exposed and naive eyes (Fig. 5; p ≤ 0.05). This data suggested that acrolein exposure disrupted collagen levels in eyelid tissues. The CD11b immunofluorescence showed significantly more inflammatory cells in acrolein-exposed eyelid tissues compared to vehicle-exposed and naïve eyes (Fig. 6a, and Fig. 6b; 3.91 ± 0.65 p ≤ 0.001 in 1-min exposure; and 3.11 ± 0.79 p ≤ 0.001).

Fig. 4.

Fig. 4.

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Hematoxylin and eosin staining of the eyelid sections of non-exposed control (A; Baseline), and acrolein-exposed (B, and C; 1-min and 3-min) rabbit tissue respectively show the tissue morphology on day 14. Acrolein vapor (1-, and 3-min.) exposure condition eyelid tissue showed a loss of epithelial cells, orbicular muscles, and meibomian glands, with distorted cellular architecture and increased pathology. n = 4 rabbit eyes in each group. Scale bar = 100 μm.

Fig. 5.

Fig. 5.

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Masson’s Trichrome staining of the eyelid sections of non-exposed (A; Baseline), and acrolein-exposed (B, and C; 1-min and 3-min respectively) rabbit tissues show the level of collagen deposition on day 14. Acrolein vapor exposure (1-, and 3-min.) in eyelid tissue shows increased collagen deposition (represented by increased blue color staining intensity). n = 4 rabbit eyes in each group. Scale bar = 100 μm.

Fig. 6a.

Fig. 6a.

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CD11b Immunofluorescence staining of the eyelid sections showing the presence of inflammatory cells in non-exposed (A and G; Baseline), and acrolein-exposed (B and E; 1-min, and C and F; 3-min respectively) rabbit tissue on day-14. The DAPI-stained nuclei show the number of cells in non-exposed (G) and acrolein vapor-exposed (H and I; 1- , and 3-min. respectively) eyelid tissues. Acrolein vapor exposure to the eyelid shows a noticeably increased number of inflammatory cells represented by CD11b + cells (white arrow) on day-14. All the experiments were independently performed using each sample in triplicates. n = 4 rabbit eyes in each group, and scale bar = 100 μm.

Fig. 6b.

Fig. 6b.

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CD11b quantification bar graph showing a significantly increased number of inflammatory cells to the eyelid tissue after acrolein vapor exposure in both 1-min and 3-min exposure conditions compared to non-exposed (Baseline) eyelid tissue. All the experiments were independently performed using each sample in triplicates. The results were presented as mean ± SD. The Student’s t-test was used for statistical analysis. ***p ≤ 0.001.

3.4. Oxidative stress and lipid peroxide evaluations

To support our postulate that acrolein’s toxicity to eyelids involves increased ROS and LPO production, commercially available ROS staining and LPO assays were used. A significantly increased 2′,7′-dichlorodihydrofluorescein levels in acrolein-exposed eyelid tissue sections compared to vehicle-exposed and naïve tissues were detected (Fig. 7; 4.27 ± 0.54 p ≤ 0.001 in 1-min exposure; and 3.42 ± 0.62 p ≤ 0.001). Likewise, acrolein-exposed eyelid tissues showed significantly increased LPO levels (3.02 ± 0.54 -fold in 1-min exposure and 3.25 ± 0.96 in 3-min exposure; p < 0.001) compared to vehicle-exposed and naïve tissues (Fig. 8) in LPO assay.

Fig. 7.

Fig. 7.

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DCF-DA staining showing the level of ROS production in non-exposed (C; Baseline), and acrolein-exposed rabbit tissue (A, and B; 1-min and 3-min) respectively at 14 days. Acrolein vapor (1-min., and 3-min.) exposed eyelid tissue-showed a noticeable amount of increase in ROS production represented by a higher amount of red color intensity as indicated by white arrows and also showed in a bar graph (D). n = 4 rabbit eyes in each group. All the experiments were independently performed using each sample in triplicates. The results were presented as mean ± SD. The Student’s t-test was used for statistical analysis. ***p ≤ 0.001. Scale bar = 100 μm.

Fig. 8.

Fig. 8.

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Acrolein exposure (1-, and 3-min.) led to increased oxidative stress lipid peroxides (LPO) in rabbit eyelid tissue compared to the naïve control (baseline) group. n = 4 rabbit eyes in each group. All the experiments were independently performed using each sample in triplicates. The results were presented as mean ± SD. The Student’s t-test was used for statistical analysis. ***p ≤ 0.001.

3.5. Evaluation of inflammatory and lipid mediators

The protein levels of an endogenous lipid mediator of inflammation, leukotriene B4 (LTB4), were also measured. Acrolein-exposed eyelid tissues showed significantly elevated LTB4 levels (1.89 ± 0.28 -fold in 1-min exposure and 2.31 ± 0.36 in 3-min exposure, p < 0.001; Fig. 9) compared to vehicle-exposed and naïve eyelid tissues. The changes in gene expression of inflammatory and lipid mediators after acrolein in eyelid tissues were studied with qRT-PCR (Fig. 10). Acrolein-exposed eyelid tissues showed significantly increased Cox-2 (3.07 ± 0.53 -fold in 1-min exposure and 3.86 ± 0.72 in 3-min exposure; p < 0.001; Fig. 10A), LOX (0.67 ± 0.20 -fold in 1-min exposure and 0.31 ± 0.07 in 3-min exposure; p < 0.001; Fig. 10B), and PAF (0.75 ± 0.05 -fold in 1-min exposure and 0.25 ± 0.09 in 3-min exposure; p < 0.001; Fig. 10C) compared to vehicle exposed and naïve eyelid tissues.

Fig. 9.

Fig. 9.

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Acrolein exposure (1-, and 3-min.) increased leukotrienes level (LTB4) in rabbit eyelid tissue compared to the naïve control (baseline) group. n = 4 rabbit eyes in each group. All the experiments were independently performed using each sample in triplicates. The results were presented as mean ± SD. The Student’s t-test was used for statistical analysis. ***p ≤ 0.001.

Fig. 10.

Fig. 10.

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Acrolein exposure (1-, and 3-min.) alters the inflammation, and lipid biosynthesis-related gene expression in rabbit eyelid tissue compared to the naïve control (baseline) group. The qRT-PCR quantification data showed the Cox-2 expression (A) significantly increased while at the same time, LOX and PAF expression (B and C) decreased significantly compared to the naïve control (baseline) group. n = 4 rabbit eyes in each group. All the experiments were independently performed using each sample in triplicates. The results were presented as mean ± SD. The Student’s t-test was used for statistical analysis. ***p ≤ 0.001.

4. Discussion

Acrolein, a volatile unsaturated aldehyde, is a strong vesicant agent. It is categorized under the threat chemical category by The Agency for Toxic Substances and Disease Registry (ATSDR) due to high toxicity and hazardous properties (Ashizawa et al., 2007; Grant WM, 1974; Kashiwagi and Igarashi, 2023; Ronald E, 2007). Acrolein showed severe pathological morbidity in many tissues including the lung, liver, cornea/eye, etc (Beauchamp et al., 1985; Dachir et al., 2015; Gupta et al., 2020, 2021; Moghe et al., 2015). Acrolein was used in World War I as a chemical warfare agent (Ghilarducci and Tjeerdema, 1995; Gupta et al., 2021). Acrolein exposure to non-ocular tissues showed a highly toxic response, morbidity, and pathological alteration (Moghe et al., 2015). In the ocular tissue, acrolein toxicity was studied sparingly and only in the corneal and retinal tissues (Alfarhan et al., 2020; Grigsby et al., 2012; Gupta et al., 2020, 2021; Murata et al., 2019). The clinical case studies found subjects with ocular pain, corneal burns, lacrimation, blepharoconjunctivitis, corneal opacity, and other ocular morbidity (Claeson and Lind, 2016; Dwivedi et al., 2015). In this study, we investigated the acrolein-mediated toxicity to the eyelid tissue and the role of lipid mediators in acrolein-induced eyelid toxicity.

Presently, no systematic data from in vivo studies are available on acrolein poisoning in the eyelid tissue most likely due to lack of suitable experimental model. We have recently optimized a customized technique utilizing a plastic vapor cup to study acrolein’s toxicity to various ocular tissues, especially the anterior segment of the eye (Gupta et al., 2020). This model imitates many features of ocular exposure of acrolein in humans as shown in Supplementary Fig. 1. We investigated acrolein’s toxicity to eyelids using a vapor-cup technique and rabbit eyes that were clinically monitored, and imaged at regular intervals to study the toxicological effects of acrolein to the eyelids. The clinical observation and stereo-microscopy identified severe inflammation, ocular pain, ocular irritation, conjunctival blepharitis, telangiectasia, and secretions on the eyelid margins of rabbits (Fig. 1). These clinical observations in rabbit eyes corroborated well with human ocular anomalies (Claeson and Lind, 2016; Dachir et al., 2015; Dwivedi et al., 2015). Claeson and Lind (2016) reported ocular irritation, lacrimation, and inflammation at the differing intensity of time- and individual variation in human volunteers who participated in investigations of acrolein toxicity. In our study, irritation, blepharitis, and inflammation in rabbits contributed to spike IOP at early time points that declines at later points (Fig. 2A and B). These observations were in agreement with the literature reporting role of eyelid closure, irritation, and inflammation in the elevation IOP resulting in glaucoma, another ocular morbidity (Jamal et al., 2002; Maul et al., 2012; van der Bosch et al., 2022).

The eyelids play a key role in protecting the eyes. The opening and closing phenomenon of the eyelids helps to spread tears over the ocular surface and keep it moist evenly (O’Neil et al., 2019). Additionally, eyelids provide a mechanical barrier against injury by closing rapidly. The acrolein ocular exposure resulted in eye irritation that may compromise the lacrimation of the tear gland. The tear flow data suggested that acrolein exposure initially triggers the tear flow secretion due to ocular irritation after chemical exposure (Fig. 3A and B). A compromised tear secretion may result in ocular epiphora and dry eye-related symptoms (Garrity, 2022; O’Neil et al., 2019; Siddireddy et al., 2018; Zhang et al., 2017).

Human and animal research showed that eyelid lipid mediators play an important role in maintaining normal visual function (Fliesler, 2010; Gilroy and Bishop-Bailey, 2019; Kojima et al., 2005). The histology data analysis of acrolein-exposed rabbit eyelid tissue sections showed the presence of macrophages, loss of epithelial cells and loss of orbicular muscles, reduced number of meibomian glands, and distorted cellular architecture (Fig. 4). Additionally, acrolein-exposed rabbit eyelid showed higher amount of collagen deposition (Fig. 5) and increased inflammatory cells (Fig. 6). The changes in collagen deposition and other compromised parameters in eyelids require detailed and rigorous investigation. This study does not address the role and functional relevance of the compromised factors in the eye after acrolein exposure, which is a limitation. Our future will address these shortcomings of the study.

Both, ocular and non-ocular tissues demonstrated a higher amount of ROS production after acrolein exposure (Ashizawa et al., 2007; Dachir et al., 2015; Dwivedi et al., 2015; Gupta et al. 2020, 2021; Moghe et al., 2015). In this study, the eyelid tissue also showed elevated ROS and LPO production (Figs. 7 and 8) after acrolein injury. The elevated ROS and LPO post-injury are linked to inflammation and morbidity of tissue, if not treated properly (Forrester et al., 2018; Kvietys and Granger, 2012; Mittal et al., 2014). Meibomian gland epithelial cells secrete an enormous amount of leukotrienes and mucus to dilute the impact of injury (Ambaw et al., 2020; Gupta et al., 2020). We noted an increased inflammation and the mRNA and protein expression of inflammatory factors (Fig. 10). We postulate that altered tear secretion and inflammatory response change lipid mediators, and tear lipid composition, and play a crucial role in acrolein-induced eyelid toxicity. Leukotrienes are known to enrich vascular permeability, spike neutrophil recruitment, and increase cytokine levels during edema and inflammation in the eye (Sahin et al., 2012). We have seen elevated levels of LTB4 in both 1- and 3-min acrolein vapor-exposed eyelids, which indicated their role in this process. However, in this study, we did not investigate the precise role of LTB4 in the pathophysiology of acrolein vapor-exposed eyelids, which is another limitation of the study.

In conclusion, acrolein exposure to the eyes caused significant morbidity to the eyelid tissues clinically and a significant increase in key inflammatory mediators, oxidative stress, and lipid mediators in rabbits in vivo (Fig. 11). Additional studies are warranted to uncover the mechanistic role of inflammatory and lipid mediators in acrolein-mediated toxicity in eyelids and ocular surface tissue functioning.

Fig. 11.

Fig. 11.

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Schematic illustration showing that acrolein exposure to the eye results in severe eyelid inflammation and cellular toxicity as a result of alteration in lipid mediators, which triggers the different ocular anomalies.

Supplementary Material

Fig 1

Acknowledgments

This work was mainly supported by the Ruth M. Kraeuchi Missouri Endowed Chair Fund (R.R.M) of the University of Missouri, Columbia, and a pilot grant (S.G.) from the Harry S. Truman VA Medical Research Foundation, Columbia, Missouri, USA. Partial support from the 5R21EY030233, 1R01EY030774, and 1U01EY031650 grant (R.R.M.) from the National Eye Institute, National Insitiute of Health, Bethesda, Maryland, USA; and 1I01BX000357 and IK6BX005646 awards (R.R.M.) from the United States Department of Veterans Affairs BLR&D, Washington DC, USA.

Footnotes

Declaration of competing interest

None of the authors have any conflict of interest to disclose.

Disclaimer

The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.exer.2023.109575.

Data availability

Data will be made available on request.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig 1
NIHMS2061973-supplement-Fig_1.tif (1.4MB, tif)

Data Availability Statement

Data will be made available on request.

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