Trehalose protects against ocular surface disorders in experimental murine dry eye through suppression of apoptosis

doi:10.1016/j.exer.2009.03.015

Experimental Eye Research

Volume 89, Issue 3, September 2009, Pages 311-318

https://doi.org/10.1016/j.exer.2009.03.015 Get rights and content

Abstract

The disaccharide trehalose is a key element involved in anhydrobiosis (the capability of surviving almost complete dehydration) in many organisms. Its presence also confers resistance to desiccation and high osmolarity in bacterial and human cells by protecting proteins and membranes from denaturation. The present study used a novel murine dry eye model induced by controlled low-humidity air velocity to determine whether topically applied trehalose could heal ocular surface epithelial disorders caused by ocular surface desiccation. In addition, the efficacy of 87.6 mM trehalose eyedrops was compared with that of 20% serum, the efficacy of which has been well documented. Mice ocular surface epithelial disorders were induced by exposure of murine eyes to continuous controlled low-humidity air velocity in an intelligently controlled environmental system (ICES) for 21 days, which accelerated the tear evaporation. The mice were then randomized into three groups: the control group received PBS (0.01M) treatment; a second group received 87.6 mM trehalose eyedrops treatment; and the third group received mice serum eyedrops treatment. Each treatment was administered as a 10 μl dose every 6 h for 14 days. The resultant changes in corneal barrier function and histopathologic examination of cornea and conjunctiva were analyzed and the level of apoptosis on the ocular surface was assessed using active caspase-3. After 14 days of treatment, the corneal fluorescein staining area, the ruffling and desquamating cells on the apical corneal epithelium, as well as the apoptotic cells on ocular surface epithelium had significantly reduced in eyes treated with trehalose compared with those treated with serum and PBS. In contrast, after 14 days of treatment, improvements in the thickness of the corneal epithelium, the squamous metaplasia in conjunctival epithelium and the number of goblet cells of the conjunctiva were less marked in eyes treated with trehalose compared with serum. These results demonstrated that trehalose could improve the appearance of ocular surface epithelial disorders due to desiccation through suppression of apoptosis. Trehalose produces some of the same responses as serum upon topical application and can maintain corneal health.

Introduction

Dry eye is associated with a decrease in tear aqueous production and an abnormality of the lipid, protein, and mucin profiles in tears. These changes result in desiccation of the ocular surface and are major causes of ocular discomfort (Lemp, 1995).

To increase the volume and improve the stability of aqueous fluid on the ocular surface, conventional dry eye management involves frequent application of preservative-free artificial tears or punctal occlusion. In addition, tear replacement therapy with autologous serum has been attempted to compensate for the insufficient composition of tear fluid along with aqueous fluid on the basis that the growth factors, cytokines, vitamins, and nutritional substances content in serum resembles aqueous tears (Tsubota and Higuchi, 2000). Fox et al. (1984) initially reported the beneficial effect of autologous serum application on dry eye in Sjögren's syndrome and the advantage of serum over artificial tears. Autologous serum application has also been reported to be effective in the treatment of severe dry eye states associated with ocular pemphigoid, Stevens-Johnson syndrome, and chronic graft-vs.-host disease (Tsubota et al., 1996, Rocha et al., 2000) and in corneas with persistent epithelial defect (Tsubota et al., 1999a). These clinical results showed that tear replacement therapy using autologous serum is an effective tool in clinical management of dry eye, and suggest that providing a proper environment for the ocular surface cells is the key to producing effective ophthalmic formulations.

The disaccharide trehalose is a key element involved in anhydrobiosis (the capability of surviving almost complete dehydration) in many organisms. (Crowe et al., 1992, Elbein et al., 2003). Its presence also confers resistance to desiccation and high osmolarity in bacterial (Leslie et al., 1995) and human cells (Guo et al., 2000) by protecting proteins and membranes from denaturation. Pretreatment with trehalose solution has been reported to protect human corneal epithelial cells in culture from death by desiccation, and a 100 mM concentration was shown to be the most appropriate dose (Matsuo, 2001). Trehalose eyedrops were also found to be a better treatment for moderate-to-severe dry eye syndrome in comparison with two commercially available eyedrops containing hyaluronan or hydroxyethylcellulose (Matsuo, 2004). These results demonstrated the potential therapeutic efficacy of trehalose in dry eye conditions. However, the precise effects of trehalose on ocular surface epithelial disorders have not been investigated.

The present study used a novel murine dry eye model induced by controlled low-humidity air velocity to determine whether topically applied trehalose could heal ocular surface epithelial disorders caused by ocular surface desiccation. In addition, the efficacy of 87.6 mM trehalose eyedrops was compared with that of 20% serum, the efficacy of which has been well documented.

Section snippets

Animals

This animal experiment was approved by the Animal Care and Ethics Committee of Wenzhou Medical College, Wenzhou, China, and adhered to the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research. A total of 60 female BALB/c mice (age range, 4–6 weeks) were supplied by the Animal Breeding Unit of Wenzhou Medical College.

Ophthalmic solutions

Trehalose (87.6 mM) preservative-free eyedrop was supplied by Laboratories Thea (Clermont-Ferrand, France).

Aqueous tear production

The phenol thread wetting was significantly reduced after 21 days of ocular surface desiccation in the ICES (from 2.3 ± 0.3 mm to 1.7 ± 0.3 mm, P < 0.01, n = 30 eyes). No significant differences were found in the test values between groups before application of eyedrops and after 7 days of treatment (1.6 ± 0.3–1.8 ± 0.4 mm for all groups, P > 0.05 for each, n = 10 eyes). However, after 14 days of treatment, both the TT and the ST groups showed a significantly greater increase in the phenol

Discussion

In this study, we used a novel murine dry eye model to evaluate the effect of trehalose eyedrops on ocular surface epithelial cell disorders. We demonstrated that trehalose, as well as serum, improves the appearance of ocular surface epithelial disorders due to ocular surface desiccation and this improvement is accompanied by reduction of apoptosis.

In this murine model, similar to human KCS, corneas of those mice raised in ICES showed reduced tear production, increased fluorescein staining,

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This was a post-hoc analysis of three clinical trials assessing the efficacy of artificial tears containing trehalose (3%) and HA (0.15%) in women with an Ocular Surface Disease Index (OSDI) ≥ 18. Patients instilled one drop of the artificial tears in each eye 3 to 6 times daily and were evaluated at baseline and after 84 ± 7 days for DED symptom severity (OSDI), hyperemia (McMonnies scale), tear break-up time (TBUT), corneal and conjunctival staining (Oxford and Van Bjisterveld scales), tear production (Schirmer I test), and ocular symptoms.
A total of 273 women were evaluated, 61 of age 42–54 years; 212 of ≥ 55 years. DED symptoms, as measured by the OSDI, decreased significantly with the treatment in both age groups (p < 0.0001). Conjunctival hyperemia decreased significantly and TBUT increased significantly in both groups, especially in women of age 42–54 (both p < 0.0001). The global (corneal and conjunctival) staining score decreased significantly in both groups, but also more in women of age 42–54 years. No differences were observed between age groups for any of the variables measured, except for visual acuity. DED symptoms were consistently reported more frequently by the mixed hormonal status women, but also the effect of the treatment was more pronounced in this group.
Artificial tears with trehalose and HA significantly improved the symptoms of DED in women aged 42–54 and ≥ 55 years. The decrease in symptoms was more pronounced in women of age 42–54 years, suggesting better mechanisms of recovery from inflammation and loss of ocular surface homeostasis.
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Eyeballs, one of the few organs exposed on the body surface, that are coated by the thin tear film to protect the ocular surface and allow clear vision [30–33], could effectively avoid the systemic clearance of therapeutic agents and provide a relatively high drug concentration on the ocular surface (i.e., in the tear fluid) via the topical ocular drug delivery (i.e., instillation). Meanwhile, the tear fluid resembles serum in many aspects and is composed of functional components such as soluble antibodies, mucous proteins, enzymes and ions [34,35]. Accordingly, there have been a number of ophthalmic drug delivery systems for the tear fluid microenvironment, including pH-sensitive, thermo-sensitive and ionic-triggered systems [30,33,36–39].
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It also protects cellular proteins from apoptosis by halting their denaturation under cold, heat and osmotic stress, oxidant injury, dehydration, and desiccation [123]. In murine models, at concentrations ranging from 30 to 200 mg/mL, 3% trehalose was proven to have a protective effect on desiccated corneal epithelial cells [123]. However, trehalose has not been formally studied in patients with NK.
Neurotrophic keratopathy (NK) is a degenerative corneal disease produced by different factors, including infection, trauma, and neurogenesis, that lead to trigeminal nerve damage and impaired corneal sensitivity. Extensive epithelial breakdown, impaired corneal epithelial healing and corneal ulceration, stromal melting, and perforation are main NK features. The proliferation of the corneal epithelium is endogenously regulated by a balance between adrenergic cAMP-dependent and cholinergic cGMP-dependent pathways. A careful balance of epitheliotropic neuromediators and neurotrophic factors expressed by corneal nerves and epithelial cells, respectively, is required to maintain corneal homeostasis. Even in its early stages, NK can cause reduced vision secondary to epithelial disturbance. Diagnosing NK is challenging, requiring the acquisition of a thorough clinical history and a comprehensive neurological and ophthalmic examination. Following suspicion of a clinical NK diagnosis, corneal sensitivity must be assessed qualitatively with the wisp of the cotton-tipped applicator and quantitatively through Cochet-Bonnet esthesiometry (CBE). A myriad of therapies is used for NK, and new, more specific modalities are being developed and investigated. Medical treatment with topical recombinant human nerve growth factor and surgical treatment through corneal neurotization are promising therapies aiming to target NK pathophysiology. Coexistent ocular surface disorders must be managed concomitantly to improve its prognosis. This review describes the up-to-date knowledge of the molecular basis regarding the pathogenesis of NK, and the novel target-specific therapeutic approaches based on this molecular mechanism.
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Moreover, it is noteworthy that oxidative stress-mediated apoptosis plays an important role in Cd-induced hepatotoxicity [5,8,11]. Many studies have demonstrated that Tr exerts its cytoprotective action through preventing apoptosis [24,25,47,48]. For example, it was proved that Tr-based eye drops is effective in the treatment of severe human dry eye by inhibiting apoptosis [49].
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^☆: Supported by Grant M303768 from the Zhejiang Provincial Natural Science Foundation of China (CW) and the Key Research Development Program Project Grant 2004C14005, Zhejiang Province, China (JQ).

¹: These authors contributed equally to this work.

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Trehalose protects against ocular surface disorders in experimental murine dry eye through suppression of apoptosis☆

Abstract

Introduction

Section snippets

Animals

Ophthalmic solutions

Aqueous tear production

Discussion

Exp. Eye Res.

Ophthalmology

Lancet

Ophthalmology

Flow cytometric analysis of inflammatory markers in conjunctival epithelial cells of patients with dry eyes

Invest. Ophthalmol. Vis. Sci.

A murine model of dry eye induced by an intelligently controlled environmental system

Invest. Ophthalmol. Vis. Sci.

Anhydrobiosis

Annu. Rev. Physiol.

Dry eye-induced conjunctival epithelial squamous metaplasia is modulated by interferon-γ

Invest. Ophthalmol. Vis. Sci.

IL-17 disrupts corneal barrier following desiccating stress

Mucosal. Immunol.

New insights on trehalose: a multifunctional molecule

Glycobiology

Beneficial effect of artificial tears made with autologous serum in patients with keratoconjunctivitis sicca

Arthritis. Rheum.

The role of apoptosis in the pathogenesis of canine keratoconjunctivitis sicca: the effect of topical Cyclosporin A therapy

Cornea

Trehalose expression confers desiccation tolerance on human cells

Nat. Biotechnol.

Albumin rescues ocular epithelial cells from cell death in dry eye

Curr. Eye Res.