# Effects of topical docosahexaenoic acid on postoperative fibrosis in an animal model of glaucoma filtration surgery

Chloé Carré, Florian Baudin, Bénédicte Buteau, Lucy Martine, Stéphane

Grégoire, Glenda Vasku, Olivier Bordeaux, Arnaud Béduneau, Yann

Pellequer, Jasmine Jamoussi, et al.

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## ► To cite this version:

Chloé Carré, Florian Baudin, Bénédicte Buteau, Lucy Martine, Stéphane Grégoire, et al. Effects of topical docosahexaenoic acid on postoperative fibrosis in an animal model of glaucoma filtration surgery. Acta Ophthalmologica Scandinavica, 2023, 101 (1), pp.e61-e68. 10.1111/aos.15222. hal-03753687

HAL Id: hal-03753687

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Distributed under a Creative Commons Attribution 4.0 International License
ORIGINAL ARTICLE

Acta Ophthalmologica

# Effects of topical docosahexaenoic acid on postoperative fibrosis in an animal model of glaucoma filtration surgery

Chloé Carré<fnref n="1" />,<fnref n="2" />

Florian Baudin<fnref n="1" />,<fnref n="3" /> ID

Bénédicte Buteau<fnref n="2" />

Lucy Martine<fnref n="2" />

Stéphane Grégoire<fnref n="2" />

Glenda Vasku<fnref n="2" />

Olivier Berdeaux<fnref n="2" />

Arnaud Béduneau<fnref n="4" />

Yann Pellequer<fnref n="4" />

Jasmine Jamoussi<fnref n="4" />

Catherine Desrumeaux<fnref n="5" />,<fnref n="6" />,<fnref n="7" />,<fnref n="8" />

Serge Aho<fnref n="9" />

Alain-Marie Bron<fnref n="1" />,<fnref n="2" /> ID

Niyazi Acar<fnref n="2" />

Catherine Creuzot-Garcher<fnref n="1" />,<fnref n="2" /> ID

Pierre Henri Gabrielle<fnref n="1" />,<fnref n="2" /> ID

Correspondence

Alain-Marie Bron, Department of
Ophthalmology, University Hospital,
Dijon, France.

Email: alain.bron@chu-dijon.fr

Funding information

Fondation de France/Fondation de l'œil;
Fond européen de développement régional;
Conseil Régional de Bourgogne, Franche-
Comté; INRAE; Agence Nationale de la
Recherche, Grant/Award Number: ANR-
11-LABX-0021-01

Abstract

**Purpose:** The aim of this study was to evaluate docosahexaenoic acid (DHA) as a potential antifibrotic agent after glaucoma filtration surgery (GFS) in rats.

**Methods:** A total of 36 10-week-old Brown Norway rats underwent GFS. Animals were equally divided into three groups: a control group, a DHA group and a mitomycin C (MMC) group. Intraocular pressure (IOP) was measured using a dynamic rebound tonometer, and a photograph of the surgical site was taken on days 1, 3, 7, 10, 14 and 17. The incorporation of DHA into fibroblasts was evaluated by gas chromatography. The expression of alfa-smooth muscle actin (α-SMA) and Smad proteins was assessed by Western blotting.

**Results:** IOP decreased after surgery in animals from the three groups on day 1 after surgery. Over time, IOP remained lower in the DHA and MMC groups than in the control group (median [interquartile range] 8.0 [7.0–8.0] and 8.0 [7.3–8.0] mmHg vs. 9.0 [8.0–9.0] mmHg, respectively; *p*<0.001). Bleb area in the DHA and MMC groups remained larger than that of the control group from day 7 to day 14 (3.9 [2.9–5.2] and 3.5 [2.3–4.4] mm<fnref n="2" /> vs. 2.3 [2.0–2.8] mm<fnref n="2" />, respectively; *p* = 0.0021). We did not observe any change in DHA concentrations in the fibroblasts of the DHA group compared with the other groups.

**Conclusion:** The impact of DHA on IOP and bleb area was similar to that of MMC. The mechanisms of action of DHA in rat eye fibroblasts deserve further investigation.

KEYWORDS

docosahexaenoic acid, fibrosis, glaucoma, glaucoma surgery

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.

© 2022 The Authors. *Acta Ophthalmologica* published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation.

<footnote n="1">¹Department of Ophthalmology, University Hospital, Dijon, France</footnote>

<footnote n="2">²Eye and Nutrition Research Group, Centre des Sciences du Goût et de l'Alimentation, AgroSup Dijon, CNRS, INRAE, Université de Bourgogne Franche-Comté, Dijon, France</footnote>

<footnote n="3">³Équipe d'Accueil (EA 7460): Physiopathologie et Épidémiologie Cérébro-Cardiovasculaires (PEC2), Faculté des Sciences de Santé, Université de Bourgogne-Franche-Comté, Dijon, France</footnote>

<footnote n="4">⁴PEPITE EA4267, Labex LipSTIC, Bourgogne-Franche-Comté University, Besançon, France</footnote>

<footnote n="5">⁵Molecular Mechanisms in Neurodegenerative Dementia Laboratory (MMDN), INSERM, U1198, Environmental Impact in Alzheimer's Disease and Related Disorders (EiAlz) Team, Montpellier, France</footnote>

<footnote n="6">⁶University of Montpellier, Montpellier, France</footnote>

<footnote n="7">⁷EPHE, Paris, France</footnote>

<footnote n="8">⁸MMDN, University of Montpellier, INSERM, EPHE, Montpellier, France</footnote>

<footnote n="9">⁹Department of Epidemiology and Biostatistics, University Hospital, Dijon, France</footnote>
# 1 | INTRODUCTION

Glaucoma is the leading cause of irreversible blindness worldwide (Jonas et al., 2017). The only proven and accepted way to lower the risk of glaucoma progression is to reduce intraocular pressure (IOP). Glaucoma surgery is one of the IOP-lowering treatment modalities available besides medical and laser therapy (European Glaucoma Society, 2020).

Trabeculectomy remains the gold standard for glaucoma surgery (Sunaric Megevand & Bron, 2021). A guarded-filtration fistula is created between the anterior chamber and the subconjunctival space to increase aqueous humour (AH) filtration and to form a bleb. However, an important complication is the development of fibrosis of the filtering bleb, which often reduces the outflow of AH through the surgically created drainage route (Cordeiro et al., 2000). Mitomycin C (MMC) is the most popular antifibrotic agent for preventing scar formation after glaucoma filtration surgery (GFS) (Chen et al., 1990). However, ocular use of MMC can lead to a variety of complications such as iatrogenic cataracts, the toxicity of corneal endothelial cells, hypotony-related maculopathy or endophthalmitis (Khaw et al., 2020; Mietz et al., 2005; Yamamoto et al., 2014). Therapeutic alternatives to MMC are then necessary to limit the development of postoperative fibrosis after GFS and to find a good balance between efficacy and safety. Indeed, the main determinant of long-term surgical success is the wound healing response (Van de Velde et al., 2015).

Docosahexaenoic acid (DHA) has been reported as a potential actor in glaucoma environment (Lafuente et al., 2021; Wang et al., 2018). DHA is a polyunsaturated fatty acid (PUFA) of the n-3 series. It is found in high concentrations in cell membranes of the outer segment of retinal photoreceptors (SanGiovanni & Chew, 2005). DHA displays anti-apoptotic and anti-angiogenic properties that have been widely documented for over 10 years (Chapkin et al., 2009; Merendino et al., 2013; Poorani et al., 2016). Antifibrotic effects have also been reported for DHA in cardiac and lung fibroblasts in rodents (Chen et al., 2011, 2013). Fibrosis lies in the differentiation of fibroblasts into myofibroblasts (Pakshir & Hinz, 2018). A typical characteristic of myofibroblasts is the expression of alpha-smooth muscle actin (α-SMA) protein following stimulation by transforming growth factor-beta (TGF-β) (Massague & Wotton, 2000). TGF-β2 represents one of the key molecular actors of fibrosis. Its action on α-SMA is driven in part by the SMAD-dependent signalling pathway (Shu & Lovicu, 2017). DHA could therefore represent an alternative to MMC used for glaucoma surgery.

The present study aimed to evaluate the antifibrotic properties of topical DHA compared to MMC in an experimental model of GFS in rats. We also attempted to characterise the underlying molecular pathway involving the α-smooth muscle actin (α-SMA) and Smad proteins.

# 2 | METHODS

All experimental animal procedures were performed in accordance with the recommendations of the Association

for Research in Vision and Ophthalmology (ARVO) and the European directives on the protection of animals used for scientific purposes (Directive 2010/63/EU). All procedures were also approved by the Ministry of Higher Education and Research and the Ethics Committee for Animal Experimentation of the Grand Campus of Dijon (#105) (APAFIS authorisation @21861-201912161022698 v1, personal authorisation n°21CAE086 for N Acar and animal facility agreement no. A21231010EA).

## 2.1 | Rat model of glaucoma filtration surgery

A total of 36 Brown Norway rats (10 weeks old; Charles River Laboratories, Lyon, France) were used. General anaesthesia was induced with isoflurane using a gas anaesthesia system (isoflurane 2% with an $O_2$ flow rate of 2 L/min; Minerve, Esternay, France). One drop of oxybuprocaine chloride (1.6 mg/0.4 ml; Thea Laboratories, Clermont-Ferrand, France) was instilled in each eye and then one drop of povidone-iodine 5% was delivered to prevent site infection (Betadine 5%; Meda Pharma Laboratories, Paris, France). Filtration surgery was performed on both eyes as described in mice by Van Bergen et al. (2015). Briefly, under a surgical microscope, the conjunctiva was surgically dissected at the limbus to expose the sclera, and a subconjunctival space was created by the surgical scissors underneath the dissected conjunctiva. Then, a 30-gauge needle was inserted through the sclera into the anterior chamber of the eye to create a fistula between the anterior chamber and the subconjunctival space. The conjunctiva was then sutured at the limbus (8-0 Ethicon Vicryl; Jonhson&Johnson, Issy-les-Moulineaux, France). One drop of a topical combination ointment containing dexamethasone and neomycin (Chibro-Cadron; Thea Laboratories, Clermont-Ferrand, France) was administered at the end of the procedure.

## 2.2 | Study groups

Rats were divided into three groups of 12 animals each: a control group, a DHA-treated group and an MMC-treated group. The animals of the control group were given the topical nanoemulsion without DHA once a day for 20 days. In the DHA group, rats received one drop per day of the topical nanoemulsion with DHA for 20 days. In the MMC group, a small surgical sponge (Beaver Visitec International, Inc. Waltham, Massachusetts, USA) soaked with MMC (0.2 mg/ml; Sigma, Saint Quentin Fallavier, France) was placed on the exposed sclera for 2 min during surgery. After removal of the sponge, the ocular surface and subconjunctival space were rinsed with 5 ml of saline solution. Rats in the MMC group did not receive any topical MMC preparation after surgery.

## 2.3 | Eyedrops preparation for control and DHA groups

Eyedrops given to the DHA and control groups were prepared through the spontaneous emulsification technique
(Jamoussi et al., 2021) for DHA (DHA group) and oleic acid (control group). Spontaneous nanoemulsion was performed by slowly pouring the oil and the surfactant mixture (previously homogenised with a Vortex® mixer) into the slightly magnetically stirred (300 rpm) ultrapure water phase. An amount of 0.140 g of oil based on DHA as triglycerides (Sigma, Saint Quentin Fallavier, France; for the DHA group) or on oleic acid as triolein (Sigma, Saint Quentin Fallavier, France; for the control group) was weighed into a 20-ml glass bottle. The DHA-based oil was sampled under nitrogen. A quantity of 0.98 g of Kolliphor HS15 and 0.140 g of glyceryl trioctanoate (Sigma, Saint Quentin Fallavier, France) was added before degassing the tube for 30 min under nitrogen. Subsequently, 10 ml of deionised water was degassed under nitrogen for 30 min in another glass vial. The lipid mixture was transferred to a water bath at 90°C and stirred under nitrogen for 6 min. A total of 6.24 g of water was then added to the lipid phase. The two immiscible phases were magnetically stirred in the water bath at the target temperature for 5 min and then stirred at room temperature for 15 min. A sterilising filtration of the preparation was carried out under a sterile laminar flow hood. Nanoemulsions were then aseptically placed in amber dropper bottles previously sterilised under nitrogen and then stored at 4°C. This method made it possible to obtain nanoemulsions of DHA at a concentration of 19 mg/ml.

## 2.4 | Measurement of intraocular pressure

IOP was measured using a dynamic rebound tonometer (Tonolab®, Icare, Vantaa, Finland). Baseline IOP was measured in both eyes under isoflurane gas anaesthesia before the procedure and on days 1, 3, 7, 10, 14 and 17 after surgery. The IOP value recorded for each eye consisted of an average of six consecutive measurements. IOP was always measured in the afternoon, between 2:00 and 4:00 p.m, taking into consideration a consistent cornea-probe distance (Rodrigues et al., 2021).

## 2.5 | Clinical evaluation of filtration blebs

An Iphone 6S (Iphone 6S, Apple, Cupertino, California, USA), fixed on a Zeiss® microscope at a magnification

of 2.5×, was used to take photographs. The photographs were recorded at a resolution of 1575×2100 pixels on days 3, 7, 10, 14 and 17 to follow the development of the bleb and the integrity of the cornea (Figure 1). A paper graduated in millimetres (Schirmer Plus, Gecis, Blois, France) was positioned next to the eye for each photograph. The area of each bleb was measured using the “contouring” function of the ImageJ software and then converted into square millimetres using the pixel measurement of the Schirmer ruler graduations with ImageJ software (ImageJ 1.52a, Wayne Rasband, National Institutes of Health, Bethesda, Maryland, USA). To ensure the reliability of the measurements, the size of the blebs was measured by two independent examiners on anonymised photographs (C.C. and A.M.B.).

## 2.6 | Isolation of tissues for ex vivo analysis

Animals were sacrificed by carbon dioxide inhalation on day 21. After enucleating both eyes, we collected the sclera and the conjunctiva at the level of the bleb. Samples were isolated and then instantly snap-frozen in liquid nitrogen. They were stored at -80°C until further analyses.

## 2.7 | Lipidomic analyses

Lipids were extracted from the sampled tissues following Folch's procedure (Folch et al., 1957). Total lipids were transmethylated using boron trifluoride in methanol according to Morrison and Smith (1964). Fatty acid methyl esters (FAMEs) were subsequently extracted with hexane and analysed via gas chromatography on GC Trace 1310 gas chromatograph (Thermo Scientific) using a CPSIL-88 column (100 mm × 0.25 mm i.d., film thickness 0.20 mm; Varian, Les Ulis, France) equipped with a flame ionisation detector. Hydrogen was used as the carrier gas (inlet pressure 210 kPa). The oven temperature was maintained at 60°C for 5 min, increased to 165°C at 15°C/min and maintained for 1 min, and then increased to 225°C at 2°C/min and finally maintained at 225°C for 17 min. The injector and the detector were maintained at 250°C. FAMEs were identified by comparison with commercial and synthetic standards. The data were processed using the EZChrom Elite software (Agilent

<figure><img src="image_3.png" /><figcaption>**FIGURE 1** Filtration bleb appearance after filtration surgery: (a) control group, (b) DHA group and (c) MMC group. The delineation of the bleb area is displayed in a, b and c</figcaption></figure>
Technologies, Massy, France) and are reported as a percentage of the total fatty acids.

## 2.8 | Expression of α-SMA protein and Smad proteins assessed by WB

Smad and α-SMA proteins were identified by Western blotting (WB). Tissues were previously treated with a modified RIPA buffer and stored at -80°C. The samples were denatured by adding 10× Laemmli buffer (Bio-Rad, Hercules, California, USA) with low glycerol concentration. Proteins were deposited in each well (20 µg for each of the experimental conditions) and then separated according to their molecular weight by migration on an SDS Stain-Free acrylamide gel (Mini-PROTEAN TGX, Stain-Free 4–15% Gels, Bio-Rad) at 70 V for 15 min, and then at 140 V for approximately 1 h. Proteins were then transferred to a 0.2-µm nitrocellulose membrane (Bio-Rad, reference 1620112) using the Trans-Blot Turbo Transfer System (Bio-Rad) in semi-dry condition, at 2.5 A and 25 V for 7 min. The non-specific sites were saturated by incubating the membrane for 1 h at room temperature in 1% PBS-Tween 5% skimmed milk. The proteins of interest were revealed by the successive incubation of a primary antibody followed by a secondary antibody. The primary antibodies specific to the protein of interest (anti-β-tubulin, Sigma, reference T8328 and anti-α-smooth muscle, Sigma, reference A2547) were diluted at 1/1000 in 1% PBS-Tween 5% skimmed milk buffer and incubated overnight at 4°C. The secondary antibody specific to the primary antibody (Polyclonal Goat anti-mouse/HRP, Dako, reference P0447), coupled to peroxidase (HRP), was diluted at 1/1000 in 1% PBS-Tween 5% skimmed milk and then incubated for 1 h at room temperature. Chemiluminescent protein detection (ECL) was performed using a ChemiDoc XRS reader with the Western Lightning® Plus-ECL Enhanced Chemiluminescence Substrate (NEL 104001EA, Perkin-Elmer). The ImageLab® v.4.0.1 software was used for the quantification of proteins of interest.

## 2.9 | Statistical analyses

Analyses were performed using Stata-15.1 software (StataCorp, College Station, Texas, USA). GraphPad Prism-7.0 software (GraphPad Software, San Diego, California, USA) was used to generate the graphics. Results are expressed as median and interquartile range (IQR). The tests were two-tailed and *p*-values of less than 0.05 were considered to be statistically significant. The distribution of the continuous variables was not normal according to the Shapiro-Wilk test. A mixed regression model was used to compare right and left eyes. The non-parametric Kruskal-Wallis test was used to compare the IOP and bleb areas of the three groups and to compare the expression of α-SMA and Smad proteins. Logistic regression was used for repeated measurements. To estimate the reliability of bleb area measurements, an intra-class correlation coefficient (ICC) was calculated.

# 3 | RESULTS

## 3.1 | Impact of topical treatment in vivo

IOP was measured at baseline before GFS. We did not observe any effect of laterality between right and left eyes (p = 0.19). In addition, baseline IOP was similar between the three groups (p = 0.86). In all three groups, we observed a decreased IOP on day 1 when compared with baseline, ranging from -13% to -32% (Figure 2). On day 17, IOP was 9.0 (8.0-9.0), 8.0 (7.0-8.0) and 8.0 (7.6-8.0) mmHg for the control, DHA and MMC groups, respectively (Figure 3). The group effect was stable over time and was characterised by a lower IOP in animals treated with DHA and MMC compared to control animals (p = 0.0001 for both) (Figure 4). There was no significant difference between IOP in the DHA and MMC groups over time (p = 0.38). Mixed regression analyses adjusted for laterality did not reveal any statistically significant variation in IOP over time (p = 0.27).

No effect of laterality was found for bleb areas (p = 0.86). On day 7, the bleb area was 3.4 (2.7-5.0), 4.6 (3.4-6.2) and 4.7 (4.0-6.3) mm² for the control, DHA and MMC groups, respectively (Figure 5). The bleb areas of the DHA and the MMC groups were significantly larger than those of the control group (p = 0.009 and p = 0.015, respectively), and the areas of these two groups were comparable on day 7 (p = 0.87). Mixed regression analyses showed that the bleb areas of the DHA and MMC groups remained larger over time compared with the control group on day 17 (p<0.0001 and p = 0.012, for the DHA vs. control group and the MMC vs. control group, respectively) (Figure 6). The ICC for the measurements

<figure><img src="image_4.png" /><figcaption>**FIGURE 2** Baseline and early postoperative intraocular pressure (IOP) on day 1 in a model of glaucoma filtration surgery in rats. **p**<0.01; ***p**<0.001. DHA, docosahexaenoic acid; MMC, mitomycin C</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**FIGURE 3** Postoperative intraocular pressure (IOP) on day 17 in a model of glaucoma filtration surgery in rats. **p**<0.001. DHA, docosahexaenoic acid; MMC, mitomycin C; NS, not statistically significant</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**FIGURE 4** Time course of intraocular pressure (IOP) in a model of glaucoma filtration surgery in rats. DHA, docosahexaenoic acid; MMC, mitomycin C; NS, not statistically significant</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**FIGURE 5** Filtration bleb areas on day 7 in a model of glaucoma filtration surgery in rats. *p* = 0.015; **p** = 0.009. DHA, docosahexaenoic acid; MMC, mitomycin C; NS, non statistically significant</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**FIGURE 6** Time course of filtration bleb areas in a model of glaucoma filtration surgery in rats. DHA, docosahexaenoic acid; MMC, mitomycin C; NS, non statistically significant</figcaption></figure>

of the bleb area was 0.944 (95% confidence interval [CI], 0.923–0.959).

## 3.2 | Impact of topical treatment ex vivo

For these analyses, we tested eight animals in each group. Gas chromatography analyses documented the relative composition of the fatty acids in the explants subjected to the different experimental conditions (Table 1). Whatever the treatment, the principal fatty acids represented were C16:0 (palmitic acid), C18:0 (stearic acid), C20:4n-6 (arachidonic acid) and C22:6n-3 (DHA). We

<table><caption>**TABLE 1** Fatty acid composition of total lipids purified from rat conjunctiva and sclera of the filtration bleb evaluated by gas chromatography (% of total fatty acids)</caption><thead><tr><th rowspan="2">PUFA</th><th>Control</th><th>DHA</th><th>MMC</th></tr><tr><th>n = 8</th><th>n = 8</th><th>n = 8</th></tr></thead><tbody><tr><td>14:0</td><td>0.4 (0.4–0.6)</td><td>0.4 (0.3–0.6)</td><td>0.4 (0.3–0.6)</td></tr><tr><td>15:0</td><td>0.2 (0.2–0.3)</td><td>0.2 (0.2–0.2)</td><td>0.2 (0.2–0.2)</td></tr><tr><td>dm16:0</td><td>1.5 (1.5–1.6)</td><td>1.5 (1.4–1.5)</td><td>1.6 (1.6–1.8)</td></tr><tr><td>16:0</td><td>17.4 (15.7–18.1)</td><td>16.8 (16.4–18.1)</td><td>17.6 (16.3–18.2)</td></tr><tr><td>16:1n-9</td><td>0.4 (0.4–0.5)</td><td>0.4 (0.4–0.5)</td><td>0.4 (0.4–0.5)</td></tr><tr><td>16:1n-7</td><td>1.3 (1.1–1.4)</td><td>1.3 (1.2–1.4)</td><td>1.2 (1.2–1.4)</td></tr><tr><td>17:0</td><td>0.4 (0.3–0.4)</td><td>0.4 (0.4–0.5)</td><td>0.4 (0.3–0.4)</td></tr><tr><td>dm18:0</td><td>2.8 (2.7–2.9)</td><td>2.8 (2.7–2.9)</td><td>2.9 (2.8–2.9)</td></tr><tr><td>dm18:1n-9</td><td>0.8 (0.7–0.9)</td><td>0.8 (0.7–0.9)</td><td>0.9 (0.9–1.0)</td></tr><tr><td>dm18:1n-7</td><td>0.6 (0.6–0.8)</td><td>0.6 (0.6–0.8)</td><td>0.7 (0.7–0.8)</td></tr><tr><td>18:0</td><td>20.5 (20.3–21.0)</td><td>20.8 (20.5–20.9)</td><td>20.4 (19.5–21.0)</td></tr><tr><td>18:1n-9</td><td>13.7 (12.6–14.0)</td><td>14.0 (13.8–14.1)</td><td>14.0 (13.7–14.4)</td></tr><tr><td>18:1n-7</td><td>3.5 (3.4–3.6)</td><td>3.7 (3.5–3.8)</td><td>3.7 (3.6–3.8)</td></tr><tr><td>18:2n-6</td><td>3.7 (3.5–4.1)</td><td>4.0 (3.7–4.1)</td><td>4.3 (3.8–4.7)</td></tr><tr><td>18:3n-3</td><td>0.1 (0.1–0.2)</td><td>0.1 (0.1–0.2)</td><td>0.2 (0.1–0.2)</td></tr><tr><td>18:3n-6</td><td>0.2 (0.1–0.2)</td><td>0.2 (0.1–0.2)</td><td>0.2 (0.2–0.2)</td></tr><tr><td>20:1n-9</td><td>0.2 (0.2–0.2)</td><td>0.2 (0.2–0.2)</td><td>0.2 (0.1–0.2)</td></tr><tr><td>20:1n-7</td><td>0.2 (0.2–0.2)</td><td>0.3 (0.2–0.4)</td><td>0.3 (0.2–0.4)</td></tr><tr><td>20:2n-6</td><td>0.3 (0.3–0.3)</td><td>0.3 (0.3–0.3)</td><td>0.3 (0.3–0.3)</td></tr><tr><td>22:0</td><td>0.2 (0.2–0.3)</td><td>0.3 (0.2–0.3)</td><td>0.3 (0.2–0.3)</td></tr><tr><td>20:3n-6</td><td>0.4 (0.4–0.4)</td><td>0.4 (0.4–0.4)</td><td>0.4 (0.4–0.5)</td></tr><tr><td>22:1n-9</td><td>0.2 (0.2–0.3)</td><td>0.3 (0.2–0.3)</td><td>0.3 (0.3–0.4)</td></tr><tr><td>20:4n-6</td><td>13.2 (12.5–13.8)</td><td>13.1 (12.6–13.5)</td><td>14.4 (13.3–15.0)</td></tr><tr><td>20:5n-3</td><td>0.3 (0.3–0.3)</td><td>0.2 (0.2–0.3)</td><td>0.2 (0.2–0.3)</td></tr><tr><td>24:0</td><td>0.5 (0.4–0.5)</td><td>0.4 (0.4–0.6)</td><td>0.5 (0.4–0.6)</td></tr><tr><td>24:1n-9</td><td>0.3 (0.2–0.4)</td><td>0.3 (0.2–0.4)</td><td>0.4 (0.3–0.4)</td></tr><tr><td>22:4n-6</td><td>1.0 (1.8–2.0)</td><td>1.8 (1.7–1.8)</td><td>1.9 (1.8–2.0)</td></tr><tr><td>22:5n-6</td><td>0.7 (0.7–0.8)</td><td>0.7 (0.7–0.7)</td><td>0.7 (0.7–0.8)</td></tr><tr><td>22:5n-3</td><td>0.6 (0.6–0.6)</td><td>0.6 (0.6–0.6)</td><td>0.6 (0.6–0.6)</td></tr><tr><td>22:6n-3</td><td>12.6 (11.3–14.3)</td><td>11.1 (10.4–12.7)</td><td>9.2 (6.8–11.1)</td></tr><tr><td>Total SFA</td><td>39.7 (39.1–40.5)</td><td>40.0 (39.5–40.9)</td><td>39.3 (39.0–40.4)</td></tr><tr><td>Total MUFA</td><td>20.1 (19.0–20.7)</td><td>20.8 (20.3–21.0)</td><td>20.7 (20.3–21.6)</td></tr><tr><td>Total PUFA</td><td>34.6 (33.9–35.6)</td><td>33.3 (32.5–35.2)</td><td>32.5 (31.7–34.3)</td></tr><tr><td>Total DMA</td><td>5.8 (5.6–5.9)</td><td>5.6 (5.5–6.0)</td><td>6.2 (6.0–6.6)</td></tr><tr><td>Total n-6</td><td>20.4 (19.6–20.9)</td><td>20.4 (19.7–21.1)</td><td>22.4 (21.0–23.0)</td></tr><tr><td>Total n-3</td><td>13.6 (12.5–15.4)</td><td>12.1 (11.4–13.5)</td><td>10.2 (7.9–12.1)</td></tr><tr><td>n-6/n-3</td><td>1.5 (1.3–1.7)</td><td>1.7 (1.5–1.8)</td><td>2.3 (1.7–3.1)</td></tr></tbody></table>

Abbreviations: DHA, docosahexaenoic acid; DMA, dimethylacetal; MMC, mitomycin C; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid.

did not observe any significant difference in DHA levels between the three groups (12.6% [11.3–14.3], 11.1% [10.4–12.7] and 9.2% [6.8–11.1] in the control, DHA and MMC groups, respectively). The ICC values for inter- and intra-observer repeatability were 0.94 (95% CI: 0.92–0.96) and 0.93 (95% CI: 0.90–0.95), respectively.
The differentiation of fibroblasts to myofibroblasts was evaluated through the expression of α-SMA protein in explants by WB. No difference in α-SMA expression was observed in animals treated with DHA or MMC compared to the control group (p = 0.51). No difference in Smad 2 phosphorylation (Figure 7a), Smad 3 phosphorylation (Figure 7b) and Smad 4 expression (Figure 7c) was observed between the three groups (p = 0.32, p = 0.55 and p = 0.88, respectively).

4 | DISCUSSION

The present study of a rat model of GFS aimed to evaluate the impact of topical DHA on myofibroblast transdifferentiation and IOP control. Our data did not demonstrate any inhibition of myofibroblast transdifferentiation through the use of topical DHA or MMC. We also found no increase in DHA concentration in the filtration bleb tissues of animals treated with DHA eye drops compared to the other groups. However, we found a lower IOP in the DHA- and MMC-treated animals and the maintenance of a larger filtering bleb over time compared to controls.

The levels of DHA measured in the explants of animals from the three groups were high and almost comparable to what is usually found in the human retina, that

is, 12%-15% of total fatty acids (Bretillon et al., 2008). Thus, they do not reflect the proportion of DHA usually found in human conjunctival fibroblasts, which is approximately 4% of total fatty acids (unpublished data). This discrepancy could be related to the fact that we also sampled deeper tissues than the conjunctiva and the sclera such as some debris of the ciliary body and the choroid underlying the surgical site. Ciliary bodies and the choroid are structures rich in DHA, that is, 10-15% of total fatty acids (Bretillon et al., 2008). Since we did not find any difference in the DHA values of the samples from the three groups after the procedure, we could assume that the DHA nanoparticles were not incorporated into the tissues despite daily instillation. This might be related either to a low dosage or to insufficient administration frequency. Indeed, knowing that one drop was equal to 30 µl, for a 19-mg/ml concentration in DHA drops, our rats received 11.4 mg of DHA, equivalent to 58.3 µM of DHA. According to preliminary in vitro studies on the effects of DHA on the viability of human Tenon's fibroblasts (HTFs), 200 µM DHA showed a dose-dependent efficacy in the migration of HTFs similar to MMC at 0.01 mg/ml (De Lazzer et al., 2017); moreover, 200 µM DHA inhibited the differentiation of fibroblasts into myofibroblasts, confirmed by the decreased α-SMA expression (De Lazzer et al., 2017). Since we could not integrate this 200-µM dose of DHA into nanovectors with the spontaneous emulsification technique (Jamoussi et al., 2021), the optimal regimen should have been increased to one drop four times a day. However, potential toxicity with a higher amount of DHA should be studied on conjunctival cells. Indeed, for further experiments, it seems mandatory to compare DHA and MMC with a similar route, a sponge carrying the active agent on ocular tissues.

The most appropriate administration route for the DHA nanoparticles after GFS remains to be determined. Bergen and colleagues have compared intravitreal (IV), subconjunctival and intracameral routes for the administration of bevacizumab in mice models of GFS. They have shown that these routes were able to equally improve surgical outcomes, such as bleb survival, after GFS although IV injection was associated with a better bioavailability (Van Bergen et al., 2015). Min et al. (2012) have described that saratin, an antifibrotic agent, in a rabbit model of GFS improved bleb survival when administered as two postoperative subconjunctival injections. The systemic administration of antifibrotics could also be an approach to explore. Indeed, myofibroblasts in conjunctival fibrosis might originate from residing cells in the eyes and circulating bone marrow-derived fibroblasts (Khaw et al., 2020). The DHA nanoparticles could then be integrated into the cells through general circulation, rather than through local use. For our study, we opted for a topical administration of DHA. This was done to evaluate the topical efficacy of DHA while avoiding the use of gas anaesthesia, which would have been necessary for the subconjunctival administration route. It was also a consideration of a therapeutic modality compatible with human clinical practice.

In the control group, IOP returned to baseline values on day 17 after GFS. This finding is consistent with

<figure><img src="image_9.png" /><figcaption>**FIGURE 7** Relative expression of Smad proteins in the sampled tissues: (a) ratio of Phospho-Smad 2 to Smad 2, (b) ratio of Phospho-Smad 3 to Smad 3 and (c) Smad 4. DHA, docosahexaenoic acid; MMC, mitomycin C; NS, not statistically significant</figcaption></figure>
the literature describing a cascade of scarring processes (Cordeiro et al., 2000). In the MMC group, the decrease in wound healing was probably the cause of a lower postoperative IOP maintenance. By inhibiting myofibroblast transdifferentiation and proliferation, MMC enables the maintenance of a bleb through a fistula in the subconjunctival space (Wolters et al., 2021). It also can re-model the ciliary body, thus leading to a decrease in AH production (Khaw et al., 1993). In the DHA group, IOP values were lower than in the control group on day 17. Despite the lack of evidence of DHA incorporation into fibroblasts as well as the absence of α-SMA expression and activation of the Smad signalling cascade, we may suspect an intrinsic effect of the carrier on fibroblasts. Indeed, the surfactant used for nanoparticle preparation, namely, Kolliphor, might display significant cellular toxicity (Le Roux et al., 2017). This agent was used in the three groups of our study. Although lipid nanocapsules (LNCs) are a promising method for the nanovectorisation of lipophilic molecules, their cellular interaction is still poorly understood. There are many hypothetical mechanisms by which Kolliphor HS15 could be cytotoxic, such as the number of hydroxystearate chains or the hydrophilicity of the head group. In addition, the small size of the head group facilitates the diffusion of Kolliphor from the micelles, and this hydrophilic molecule could modify the cell membrane. Further investigations on the cytotoxicity of these nanovectors are needed (Bunchongprasert & Shao, 2020).

In our trial, we failed to elucidate the pathways of the antifibrotic effect of topical DHA in a GFS model in rats. Fibrosis is a crucial issue to consider, as it determines the hydraulic conductivity of the conjunctiva, which in turn controls the fluid flow through the tissue (Khaw et al., 2020).

We acknowledge several limitations in this study. First, the size of the groups was not sufficient to perform all the experiments on a single animal, which explains why only eight sampled tissues were tested for ex vivo analyses. Second, we could not determine a correlation between IOP and the bleb area because of a lack of statistical power due to the small population size. Third, our pilot study failed to decipher the potential mechanisms involving DHA in the modulation of fibrosis after GFS. Fourth, we used a GFS model in rats without prior IOP elevation and thus we were dealing with a narrow range of IOP. Further studies with a rodent model of induced glaucoma would be interesting and could show a greater variability between the groups (Biswas & Wan, 2019). Fifth, for reaching a valid lipidic analysis from a technical point of view, we included some scleral tissue to have a sufficient amount of tissue for each eye, which should be considered a limitation of our study. Finally, one should be cautious when extrapolating the results from animal research to humans. The strengths of our study were, first, the design of the trial based on a validated GFS model in rodents (Van Bergen et al., 2015). Second, the IOP measurements with gas anaesthesia limited intra- and inter-individual IOP variations and the animals' stress even though it is possible in some strains to measure IOP in the conscious rat (Cabrera et al., 1999). Indeed, isoflurane induces

minimal IOP changes (Ding et al., 2011). Third, the good inter- and intra-ICC (0.94 and 0.93, respectively) values were an indication of the acceptable repeatability of the bleb area measurements.

In conclusion, our trial was a preliminary study testing the effects of DHA eye drops on IOP modulation and filtration bleb scarring in an animal model of GFS. Further investigations are needed to find the best route of administration, the best regimen, and the best galenics for evaluating the antifibrotic effect of DHA in GFS.

## ACKNOWLEDGEMENTS

The authors thank Patrick Cailler and Laurence Decocq for animal care and Marissa Vernot-Jackson for her help in vivo experiments.

## FUNDING INFORMATION

This work was funded by Agence Nationale de la Recherche [ANR-11-LABX-0021-01]; INRAE; French "Investissements d'Avenir" Program; Conseil Régional de Bourgogne, Franche-Comté [PARI grant]; FEDER (European Regional Development Fund) and Fondation de France/Fondation de l'œil.

## CONFLICT OF INTEREST

Dr Bron reports consulting for Aerie, Allergan SAS, Bausch & Lomb, Santen and Théa. Dr Creuzot-Garcher reports consulting for Allergan SAS, Bausch & Lomb, Bayer HealthCare, Horus Pharma, Novartis Pharma SAS, Roche and Théa. No other disclosures are reported.

## ORCID

Florian Baudin https://orcid.org/0000-0003-4976-9876

Alain-Marie Bron https://orcid.

org/0000-0002-7265-931X

Catherine Creuzot-Garcher https://orcid.

org/0000-0002-3587-4598

Pierre Henri Gabrielle https://orcid.

org/0000-0002-9688-4845

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**How to cite this article:** Carré, C., Baudin, F., Buteau, B., Martine, L., Grégoire, S. & Vasku, G. et al. (2023) Effects of topical docosahexaenoic acid on postoperative fibrosis in an animal model of glaucoma filtration surgery. *Acta Ophthalmologica*, 101, e61–e68. Available from: https://doi.org/10.1111/ aos.15222