Transgenic dry eye mouse models: powerful tools to study dry eye disease

INTRODUCTION

Dry eye disease (DED) is one of the most common chronic multifactorial ocular surface diseases in the department of ophthalmology. The prevalence of DED is 5%-50% on a global scale, which is expected to rise with the aging of the population. Besides, the widespread use of digital products makes DED a much more common disorder in young generations. DED results in several ocular discomforts, vision fluctuation, and even potential damage to the ocular surface.Long term interventions will be needed once DED is diagnosed,which may substantially affect patients' quality of life. In the meantime, the decline of the workplace productivity will have a great negative impact on the economy. Overall, DED brings heavy burdens both on individuals and the society,which becomes an increasingly critical health issue.

The Tear Film and Ocular Surface Dry Eye Workshop (TFOS DEWS) II classified DED into two categories: aqueousdeficiency dry eye (ADDE) and evaporative dry eye (EDE).ADDE results from lacrimal diseases or dysfunction, which can be further divided into Sj?gren syndrome dry eye (SSDE)and non-Sj?gren syndrome dry eye (NSSDE). EDE can be divided into intrinsic and extrinsic EDE according to the etiology, and the former one mainly refers to meibomian gland dysfunction (MGD). The pathogenesis of DED is complicated and remains elusive. The fundamental mechanism of DED in all cases is the vicious circle caused by tear film hyperosmolarity and ocular surface inflammation. Tear film hyperosmolarity activates mitogen-activated protein kinase(MAPK) and NF-κB signal pathway on the ocular surface and induces innate and adaptive immune response. The elevation of interleukin-1 (IL-1), tumor necrosis factor α (TNF-α)and matrix metalloproteinase 9 (MMP9) leads to the loss of conjunctival goblet cells and subsequent decline of mucins on the conjunctiva and in the tear film. This compromises the tear film stability, and tear film instability, in turn, amplifies tear film hyperosmolarity. Henceforth, the vicious circle is generated. With the study of new pathological pathways,novel drugs that specifically target certain aspects of DED are under exploration.

At present, the treatment for DED is nonspecific, including lifestyle improvement, artificial tears, physical devices,and topical anti-inflammatory agents. The translations of new therapies first rely on suitable animal models to obtain primary efficacy and safety data. There have been many methods to establish dry eye animal models by far, including lacrimal gland excision (LGE), desiccating environmental stress (DES), genetic modification and so on. The dry eye animal models established by different methods simulate different mechanisms of dry eye subtypes and have their own advantages and limitations. Specifically, transgenic and gene knockout models are very suitable to study the mechanism of a particular pathway in the development of DED.

Mice have been greatly preferred and widely used in basic research not only for their small body size, easy operation, and relatively low cost, but also genetic and pathophysiological similarities to humans. Most transgenic mouse models are developed by promoting, suppressing, or changing the expression of a certain gene. These manipulations help to study the structure, function, and expression regulation of this gene. The etiology and pathogenesis of DED are still not fully understood. Transgenic mouse models will certainly be of much help in exploring the mechanisms of DED. Besides,they can also be reliable workhorses of novel drug screening.On one hand, their inherent features similar to human DED leave out the difficult and laborious modeling process. On the other, the identified pathogenesis of a certain model helps to investigate the specific pharmacodynamics and action mechanism of the drug. Therefore, transgenic dry eye mouse models can be very powerful tools in DED research, and it's necessary to look into and make the best of them.

This review aims to summarize existing common transgenic dry eye mouse models, and evaluate their characteristics,advantages, and limitations, trying to provide a reference for the establishment of reliable transgenic dry eye mouse models for future research work.

SJ?GREN SYNDROME DRY EYE TRANSGENIC MOUSE MODELS

Sj?gren syndrome (SS) is an autoimmune disorder characterized by dry eye (keratoconjunctivitis sicca) and a dry mouth (xerostomia), which is usually accompanied by rheumatoid arthritis, lupus and other autoimmune disorders.Many autoimmune animal models resembling SS have been developed so far. They have similar characteristics of autoimmune exocrinopathy, secretory dysfunction, female predominance and presence of autoantibodies, along with other unique features.

Natural Mice Strains with Features of Dry Eye Disease

Non-obese diabetic (NOD)mouse is a spontaneous type I diabetes (TID) mouse model.NOD mouse has been commonly utilized as a secondary SS mouse model due to its spontaneous SS-like symptoms.

Although diabetes and sialoadenitis are more prevalent in females, the onset of dacryoadenitis in male NOD mice is surprisingly higher.

A great number of studies about DED drug development have been conducted with NOD mice. The efficacy of cyclosporine A (CsA) and the drug carrier development to improve its bioavailability have been studied using NOD mice. In addition, Cathepsin S (CTSS) inhibitors and rapamycin could mitigate the ocular manifestations of SS in NOD mice.

However, the coexisting TID in NOD mice requires longterm insulin injections, which not only add the feed cost, but also potentially affect the study of SS. To overcome these problems, several NOD strains have emerged. They exhibit aqueous tear deficiency equivalent to NOD mice without TID manifestations.

NOD.B10-H2mice exhibited saliva and tear secretory loss.Focal lymphocytic infiltration was presented in the lacrimal glands (LG) of NOD.B10-H2mice. The expression levels of pro-inflammatory cytokines in the conjunctiva and LG increased with age. Additionally, novel SS autoantibodies like anti-CA6, anti-SP1 (but not anti-PSP) increased in the pre-clinical phase before the presence of antinuclear antibody(ANA).

堅持用習近平新時代中國特色社會主義思想武裝干部頭腦，教育引導黨員干部牢固樹立“四個意識”，堅定“四個自信”。堅決維護習近平總書記的核心地位，堅決維護黨中央權威和集中統一領導，在急難險重任務中加強政治訓練，確保系統內干部政治上絕對可靠、對黨絕對忠誠，確保各項工作始終沿著正確政治方向前進。

除酰胺外，脂肪酸（或甘油酯）與乙二胺等還可以在氧化鋁的催化下高溫進一步脫水環化生成咪唑啉[11]化合物，如：

1)NOD.B10-H2mice

Many novel formulations have been tested on this strain,such as high-mobility group box 1 and IRT5 probiotics.Besides, the efficacy tests of poly(ethylene glycol) and catechin nanocomplex, sulglycotide, RGN-259, and cevimelines were conducted in NOD.B10-H2mice treated with desiccation stress.

2)C57BL/6.NOD-mice

Idd3 and Idd5 are two chromosomal intervals closely correlated with the spontaneous development of SS in NOD mice. C57BL/6.NOD-mice are descended from C57BL/6.NODmice carrying() and C57BL/6.NODmice carrying().

C57BL/6.NOD-mice showed increased expression level of IL-1β, TNF-α, and declined goblet cell density in the conjunctiva at 12wk. They also exhibited lymphocytic infiltration consisting predominantly of CD4+ T cells in the LG at 20wk. Notably, the LG infiltration did not result in loss of tear production.

C57BL/6.NOD-mice were widely used to explore potential therapeutic targets of SS. Interestingly, they were more frequently seen in SS studies focused on sialadenitis.More studies focused on dacryoadenitis would be needed to testify their applicability to study DED.

3)NOD.-/- mice

針對現行的評估標準，其中有很多超過了十年的行業標準，還有很多超過了五年的國家標準，相關部門就要嚴格的按照當前我國輻射環境管理方面的法律規章制度、準則以及最新的檢測數據，對其進行重新的評估，并做好修訂工作。不僅如此，我們還要將這一項工作列入到環境保護標準年度工作規劃當中，針對當前已經無法滿足現行技術的標準，要及時的采取有效措施進行解決。最后，我們還要加大個和國際之間的交流與合作，充分的掌握更多先進的監測技術，密切關注輻射環境監測工作動態，實現和國際之間的接軌，促進我國輻射環境監測的進一步發展。

Unlike other SS models, male BXSB/MpJ-Yaa have more severe autoimmune symptoms than females because of the Y-linked autoimmune acceleration (Yaa) mutation on the Y chromosome(Table 1).

The importance of IL-4-STAT6 signal transduction pathway was investigated using NOD.-/-, NOD.B10-H2-/-, and NOD.B10-H2.C--/- mouse model. IgG antibodies against muscarinic receptor 3 (M3R) were detected in NOD.B10-H2.C-+/+ mice but not in NOD.B10-H2.C--/- mice.This suggested that IL-4-STAT6 pathway might be a potential therapeutic target for SS treatment.

4)NOD.--/-mice

Interferon-γ (IFN-γ) is indispensable in both the early and later immune phase of DED. The high expression of IFN-γ in the conjunctiva is correlated with goblet cell loss and mucin decrease.

IFN-γ increased markedly both in the LG and in tears of male NOD mice, who spontaneously developed dacryoadenitis but not sialoadenitis. However, neither the NOD.--/- nor the NOD.--/- mice exhibited leucocytic infiltration or secretory dysfunction in the LG and salivary glands (SG).Detectable antibodies were also absent. These findings suggested that IFN-γ was indispensable in the development of SS.

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MRL/+ and MRL/lpr are two congenic substrains of MRL/MpJ mice. Both MRL/+ and MRL/lpr mice develop lacrimal inflammation, but it's earlier,more severe and extensive in MRL/lpr mice.

Apart from systemic lupus erythematosus (SLE) symptoms,the phenotype of MRL/lpr mice encompasses the key features of SS, including female sex predilection, decreased saliva and tear secretion, lymphocytic infiltration in the SG and LG, and anti-SSA and anti-SSB antibodies production. Sex-related differences in gene expression contributed to the development of dacryoadenitis, and could be influenced by testosterone treatment. Increased levels of pro-inflammatory cytokines were observed in the LG. And the gene expression levels of MMP2 and MMP9 also increased. Besides, injection of p38-MAPK inhibitor, SB203580, into the LG significantly improved tear production. This indicated that the activation of p38-MAPK pathway played an important role in the development of DED of the MRL/lpr mouse model.

However, MRL/lpr mice spontaneously develop SLE-like autoimmune injuries. They're likely to die of renal failure with an average life span of 6mo. MRL/+ mice usually live up to 2y,but they are rarely used due to the late onset and slight degree of the DED.

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