Research Article: Research on the Stability of a Rabbit Dry Eye Model Induced by Topical Application of the Preservative Benzalkonium Chloride

Date Published: March 16, 2012

Publisher: Public Library of Science

Author(s): Chaoyang Li, Yiyue Song, Shaohong Luan, Pengxia Wan, Naiyang Li, Jing Tang, Yu Han, Cuiju Xiong, Zhichong Wang, Michael E. Boulton.


Dry eye is a common disease worldwide, and animal models are critical for the study of it. At present, there is no research about the stability of the extant animal models, which may have negative implications for previous dry eye studies. In this study, we observed the stability of a rabbit dry eye model induced by the topical benzalkonium chloride (BAC) and determined the valid time of this model.

Eighty white rabbits were randomly divided into four groups. One eye from each rabbit was randomly chosen to receive topical 0.1% BAC twice daily for 2 weeks (Group BAC-W2), 3 weeks (Group BAC-W3), 4 weeks (Group BAC-W4), or 5 weeks (Group BAC-W5). Fluorescein staining, Schirmer’s tests, and conjunctival impression cytology were performed before BAC treatment (normal) and on days 0, 7, 14 and 21 after BAC removal. The eyeballs were collected at these time points for immunofluorescence staining, hematoxylin and eosin (HE) staining, and electron microscopy. After removing BAC, the signs of dry eye in Group BAC-W2 lasted one week. Compared with normal, there were still significant differences in the results of Schirmer’s tests and fluorescein staining in Groups BAC-W3 and BAC-W4 on day 7 (P<0.05) and in Group BAC-W5 on day 14 (P<0.05). Decreases in goblet cell density remained stable in the three experimental groups at all time points (P<0.001). Decreased levels of mucin-5 subtype AC (MUC5AC), along with histopathological and ultrastructural disorders of the cornea and conjunctiva could be observed in Group BAC-W4 and particularly in Group BAC-W5 until day 21. A stable rabbit dry eye model was induced by topical 0.1% BAC for 5 weeks, and after BAC removal, the signs of dry eye were sustained for 2 weeks (for the mixed type of dry eye) or for at least 3 weeks (for mucin-deficient dry eye).

Partial Text

Dry eye is a common disease worldwide [1]. Clinical epidemiological surveys have demonstrated that in 1995, 17% of the outpatients in Japan reported symptoms of dry eye [2], and in 1997, 14.6% of the U.S. population who ranged in age from 65 to 84 years showed symptoms of dry eye [3]. In 2003, approximately 33.7% of the elderly population of Taiwan (those over the age of 65 years) also reported symptoms of dry eye [4]. The high prevalence of dry eye has already evoked the close attention of scholars from all over the world, so there has been a great advancement in dry eye research over the last decade. Dry eye has been redefined in a report from the Dry Eye WorkShop (DEWS), which summarized the results of these studies. Increased osmolarity of the patient’s tears and inflammation of the ocular surface were added to the new definition, and the potential damage to the ocular surface was clearly indicated [5]. However, there is still much to be learned from the study of the dry eye disease because dry eye is a multifactorial and complex disease.

No baseline (normal) significant differences were found among the four experimental groups.

Inflammation and increased tear osmolarity have been proposed as specific new additions to the definition of the dry eye disease that was reported by the Dry Eye WorkShop (DEWS) because the research regarding dry eye disease that has been conducted in recent years shows that the core pathogenesis mechanisms of dry eyes included heightened tear osmolarity and an unstable tear film [5]. According to this mechanism of pathogenesis, some symptoms of dry eye disease are due to increased tear osmolarity that arises from various causes and that can spur the ocular surface epithelial cells to produce and release inflammatory mediators into the tear film. This in turn leads to an inflammatory cascade that results in apoptosis of the ocular surface epithelial cells, and reductions in goblet cell density reducing [12], [24]. Another hypothesis regarding the pathogenesis of dry eye is one in which an unstable tear film that could be mainly caused by a mucin deficiency on the ocular surface is the initial problem, which implies that an increase tear osmolarity is not responsible for the onset of dry eye symptoms. Clinical examples of the latter include dry eye that results from the local administration of drugs that contain BAC [25], and these provide an important clinical basis of establishing this dry eye model. BAC is capable of destroying the tight junctions that are located in the superficial cells of the corneal epithelium [26]; this degradation of tight junctions then damages the normal functioning of the corneal epithelium [27]. Exposure to BAC can also lead directly to both apoptosis of the conjunctival epithelium [28], [29] and a decrease in the number of goblet cells [30]. Thus, as previous research has shown, the tear film becomes unstable and ceases to play its role in protecting the ocular surface [31]. In addition to having this effect on the cells themselves, BAC can stimulate the overexpression of inflammatory mediators in the epithelium, such as ICAM-1, or interleukins IL-6, IL-8, and IL-10 [32], [33], which may also cause apoptosis of both epithelial cells and goblet cells. The loss of these cells then leads to a reduction in mucin expression [34], [35]. These studies shows that in addition to the direct effect that BAC has on goblet cell density, inflammation underlies decreases in mucin expression. Reductions in the production of mucin speed up the rate at which the tear film breaks up, thereby aggravating the ocular surface damage and stimulating the inflammation cascade of the ocular surface epithelial cells. Ultimately, this results in a vicious cycle based on the core mechanisms of ocular surface protection that eventually leads to the occurrence of dry eye disease. A previous study demonstrated that the damage to the ocular surface that resulted from BAC administration was both dose- and time-dependent. The expression levels of apoptotic markers increased, and corneal and conjunctival cells were damaged immediately after in vitro exposure to a solution containing 0.1% BAC [35]. BAC has a high affinity for binding to ocular cells, and it can combine with the cellular plasma membranes quickly and persistently, which then results in a series of cellular alterations. At the same time, one drop of BAC can remain on the ocular surface for as long as 48 hours [36]. Furthermore, the topical application of a 0.1% solution of BAC did not cause severe damage to the ocular surface, such as ulcerations, large epithelial defects, or neovascularization. We had already constructed a rabbit dry eye model using a topical application of a 0.1% solution of BAC that was administered for 2 weeks [17]. In follow-up experiments that were based on this model, we found that the animal model that was established in this manner lacked stability, which was probably because the administration period was not long enough to cause permanent changes, so the eye that experienced the symptoms of dry eye was able to recover quickly. To obtain a stable animal model of dry eye that could be used for subsequent research, we designed the present experiment. We repeated the previous procedures that had been used to generate various models. After the cessation of BAC treatment, we observed both the self-recovery of the ocular conditions of the experimental animals and some related indices. Our results showed that the values of the indices that we measured in each experimental group on day 0 after we stopped administering the medication were consistent with the characteristics of dry eye. This finding also agreed with the results of our previous study [17] and those of studies that were conducted by other researchers [10], [15]. However, although some indicators of dry eye persisted as the time over which we continued to observe the model lengthened, some indices of the dry eye model that we had established gradually recovered.