液氮冷冻眼睑缘致萎缩性睑板腺功能障碍-动脉网

Atrophic meibomian gland dysfunction induced by eyelid margin cryotherapy with liquid nitrogen

Nature 等信源发布 2025-01-04 16:26

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可切换为仅中文

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Abstract

摘要

To develop an atrophic Meibomian Gland Dysfunction (MGD) animal model via liquid nitrogen cryotherapy, the eyelid edges of C57 mice exposure to liquid nitrogen for 30 s. Morphology of MG and ocular surface were assessed using stereomicroscopy and a slit lamp microscope at multiple time points post-injury.

为了通过液氮冷冻疗法开发萎缩性睑板腺功能障碍（MGD）动物模型，将C57小鼠的眼睑边缘暴露于液氮30秒。在损伤后的多个时间点，使用立体显微镜和裂隙灯显微镜评估MG和眼表的形态。

Acinar loss and atrophy were observed from day 7, with increased inflammation and apoptosis, and decreased proliferation in acinar cells. Corneal epithelial defects appeared after day 14. Liquid nitrogen induced selective damage to meibomian acinar cells, simulating MGD pathology effectively, with peak effects at day 21, providing a relevant model for atrophic MGD research..

从第7天开始观察到腺泡丢失和萎缩，炎症和凋亡增加，腺泡细胞增殖减少。第14天后出现角膜上皮缺损。液氮诱导睑板腺泡细胞选择性损伤，有效模拟MGD病理，在第21天达到峰值，为萎缩性MGD研究提供了相关模型。。

Introduction

简介

Meibomian glands (MGs) are sebaceous glands located within the tarsal plates of the eyelids, responsible for secreting lipids that constitute the superficial layer of the tear film

睑板腺（MGs）是位于眼睑睑板内的皮脂腺，负责分泌构成泪膜表层的脂质

. These lipids are critical for preventing tear evaporation and protecting the ocular surface from microbial and particulate matter. Meibomian gland dysfunction (MGD) is a prevalent chronic condition characterized by obstruction of the gland ducts and/or qualitative and quantitative alterations in lipid secretion.

这些脂质对于防止泪液蒸发和保护眼表免受微生物和颗粒物的影响至关重要。睑板腺功能障碍（MGD）是一种普遍的慢性病，其特征是腺管阻塞和/或脂质分泌的定性和定量改变。

. The overall global prevalence rate is reported to be between 21.2% and 71.0%

。据报道，全球总体患病率在21.2%至71.0%之间

. MGD is widely recognized as a leading cause of dry eye syndrome

MGD被广泛认为是干眼症的主要原因

. Based on the secretion status of the meibomian glands, Meibomian Gland Dysfunction (MGD) is divided into two major categories: hyposecretion type and hypersecretion type. The hyposecretion type is further subdivided into c type and obstructive type

根据睑板腺的分泌状态，睑板腺功能障碍（MGD）分为两大类：分泌不足型和分泌过多型。分泌不足型进一步细分为c型和阻塞型

Acini atrophy represents a degenerative change in the meibomian glands and is a significant pathological characteristic of Meibomian Gland Dysfunction (MGD), manifesting as a reduction in acinar cell volume and a decline in the secretion capacity of meibum

腺泡萎缩是睑板腺的退行性改变，是睑板腺功能障碍（MGD）的重要病理特征，表现为腺泡细胞体积减少和睑板分泌能力下降

. The terminal ducts of the meibomian glands may not exhibit significant obstruction, whether this is accompanied by ductal atrophy requires further research. Atrophic type can be categorized into primary and secondary types

睑板腺的末端导管可能没有明显的阻塞，是否伴有导管萎缩需要进一步研究。萎缩型可分为原发性和继发性

. The causes and mechanisms of primary meibomian gland atrophy are not well understood. Advanced age is a possible risk factor for primary gland atrophy. Type 2 diabetes, prolonged use of video display terminals, high sugar and high-fat diet, long-term use of retinoids, and long-term wear of contact lenses can lead to gland atrophy.

.原发性睑板腺萎缩的原因和机制尚不清楚。高龄可能是原发性腺体萎缩的危险因素。2型糖尿病、长时间使用视频显示终端、高糖和高脂肪饮食、长期使用类维生素A以及长期佩戴隐形眼镜可导致腺体萎缩。

. Additionally, this type of MGD can also be secondary to the increased glandular pressure caused by obstructive MGD. The ectodermal dysplasia syndrome can cause congenital developmental disorders of the meibomian gland acini, and such meibomian gland lesions do not belong to MGD

此外，这种类型的MGD也可能继发于阻塞性MGD引起的腺体压力增加。外胚层发育不良综合征可引起睑板腺腺泡的先天性发育障碍，这种睑板腺病变不属于MGD

Despite the recognized importance of MGs in ocular health, the development of reliable animal models for atrophic MGD has been a subject of ongoing debate and refinement. Various knockout mice can cause changes in the structure and function of the meibomian glands

尽管公认MGs在眼部健康中的重要性，但开发可靠的萎缩性MGD动物模型一直是一个持续争论和改进的主题。各种基因敲除小鼠可引起睑板腺结构和功能的改变

. MGD models were created by targeting the deletion of specific genes that encode enzymes or lipoproteins involved in lipid metabolism, such as High-fat diet (HFD) mice, Fatp4 − /−;Tg(IVLFatp4) mice, ELOVL3-ablated (E3hom) mice, ACAT-1-/- mice and so on

MGD模型是通过靶向缺失编码参与脂质代谢的酶或脂蛋白的特定基因而创建的，例如高脂饮食（HFD）小鼠Fatp4-/-；

. Nevertheless, since the models were developed through systemic gene knockout, we cannot discount the potential for systemic effects on the anatomy and physiology of other ocular surface tissues. For example, ACAT-1-/- mice can lead to abnormalities in adrenal gland structure, atherosclerosis of the arteries, and severe alterations in other organs.

然而，由于这些模型是通过系统性基因敲除开发的，因此我们不能忽视对其他眼表组织的解剖学和生理学产生系统性影响的可能性。例如，ACAT-1-/-小鼠可导致肾上腺结构异常，动脉动脉粥样硬化以及其他器官的严重改变。

If corneal epithelial damage occurs in a knockout animal model, it cannot be ruled out whether the gene defect itself affects the development of the cornea or whether it is a secondary change in the cornea caused by meibomian gland dysfunction. Moreover, the vast majority of patients with meibomian gland atrophy are not caused by genetic defects.

如果在基因敲除动物模型中发生角膜上皮损伤，则不能排除基因缺陷本身是否影响角膜的发育，或者它是否是由睑板腺功能障碍引起的角膜继发性变化。此外，绝大多数睑板腺萎缩患者不是由遗传缺陷引起的。

Clinical studies report that during the aging process, meibomian glands (MG) undergo acinar atrophy, thickening of the basement membrane, and inflammatory changes.

临床研究报道，在衰老过程中，睑板腺（MG）经历腺泡萎缩，基底膜增厚和炎症变化。

. Nien and colleagues examined the MG of mice at 2, 6, 12, and 24 months of age, finding that in older mice, there were alterations in the PPARγ receptor signaling, decreased cell proliferation, reduced lipid synthesis, and glandular atrophy

Nien及其同事检查了2、6、12和24个月大的小鼠的MG，发现在老年小鼠中，PPARγ受体信号发生了改变，细胞增殖减少，脂质合成减少和腺体萎缩

. These findings are consistent with age-related changes in human MG. The atrophy of MG in aged mice may be attributed to the loss of meibomian gland progenitor cells, rather than excessive ductal keratinization and gland obstruction. Therefore, aged mice can serve as a model for age-related MGD, with many similar age-related changes between mice and humans.

这些发现与人类MG的年龄相关变化一致。老年小鼠MG的萎缩可能是由于睑板腺祖细胞的丢失，而不是过度的导管角化和腺体阻塞。因此，老年小鼠可以作为与年龄相关的MGD的模型，在小鼠和人类之间具有许多类似的与年龄相关的变化。

However, the long breeding cycle of aged mice, high mortality rate, and the low rate of model development are need to considerate. The existing animal models of MGD induced by injury cause mechanical obstruction such as keratinization of the duct orifice, and do not represent the characteristics of atrophic MGD.

然而，老年小鼠的繁殖周期长，死亡率高，模型开发率低，需要考虑。现有的由损伤引起的MGD动物模型会导致机械性阻塞，例如管口角化，并且不代表萎缩性MGD的特征。

. Animal models of primary meibomian gland atrophy remain missing.

.原发性睑板腺萎缩的动物模型仍然缺失。

Temperature is crucial to the functionality of the meibomian glands, particularly as it significantly influences the physicochemical properties of the meibum secreted by these glands

温度对睑板腺的功能至关重要，特别是因为它会显着影响这些腺体分泌的睑板的理化特性

Experiments conducted using a Langmuir trough have revealed that human meibomian lipids are characterized by their ability to form multilamellar liquid crystalline films that are highly compressible and resistant to collapse. These lipids possess intrinsic surfactant properties, enabling them to effectively spread across an aqueous substrate.

使用朗缪尔槽进行的实验表明，人睑板脂质的特征在于它们能够形成高度可压缩且抗塌陷的多层液晶膜。这些脂质具有固有的表面活性剂特性，使它们能够有效地扩散到水性底物上。

However, temperature is a significant modulating factor; a decrease in temperature has been shown to augment the surfactant capabilities of these lipids.

；温度降低已被证明可以增强这些脂质的表面活性剂能力。

.The temperature at which phase transition occurs was markedly elevated by 4 degrees Celsius for the meibomian gland lipids in comparison to the mean values from age-matched controls without a history of dry-eye symptoms

。与没有干眼症病史的年龄匹配对照组的平均值相比，睑板腺脂质发生相变的温度明显升高了4摄氏度

. Patients with meibomian gland dysfunction exhibit a reduction in the surface temperature of the tarsal conjunctiva

睑板腺功能障碍患者的睑板结膜表面温度降低

. A study examines the influence of eyelid temperature on the secretion of oil from the meibomian glands. The study found that increasing the eyelid temperature enhanced oil secretion, while decreasing it reduced secretion. The results suggest that the viscosity of the meibomian oil is likely affected by temperature changes, which supports the use of warm compresses to improve oil delivery in cases of gland dysfunction.

一项研究调查了眼睑温度对睑板腺油脂分泌的影响。研究发现，提高眼睑温度可以促进油脂分泌，而降低眼睑温度可以减少油脂分泌。结果表明，睑板油的粘度可能受到温度变化的影响，这支持在腺体功能障碍的情况下使用温热的按压来改善油的输送。

. Thermo-Transient Receptor Potential (TRP) channels are detectably expressed within meibomian glands (MGs) at both the genetic and protein levels. The potential functions of TRPV1 and TRPM8 may involve the regulation of lipid metabolism in the meibomian glands (MG)

热瞬变受体电位（TRP）通道在睑板腺（MGs）中在遗传和蛋白质水平均可检测到表达。TRPV1和TRPM8的潜在功能可能涉及睑板腺（MG）脂质代谢的调节

In dermatology, cryotherapy is applied to specifically target and destroy sebaceous glands in the skin as a treatment for seborrheic dermatitis. Solid carbon dioxide (at − 78 °C) and liquid nitrogen (at − 195 °C) have long been utilized for the nonspecific ablation of epidermal lesions, including conditions like actinic keratosis and common warts.

在皮肤病学中，冷冻疗法用于特异性靶向和破坏皮肤中的皮脂腺，作为脂溢性皮炎的治疗方法。。

. They were examined in both murine and porcine models across a range of low temperatures, with subsequent trials on human participants. In the murine model, the apex of histological damage to the ears was observed at 72 h post-cooling application. It was characterized by eosinophilic necrosis within the sebaceous glands and a sustained reduction in gland count for up to seven days after treatment.

。在一系列低温下，在鼠和猪模型中对它们进行了检查，随后对人类参与者进行了试验。在小鼠模型中，在冷却后72小时观察到耳朵组织学损伤的顶点。其特征是皮脂腺内嗜酸性粒细胞坏死，治疗后长达7天的腺体数量持续减少。

The cooling process led to the disruption of Sebaceous gland cell membranes, a decrease in alkaline phosphatase activity, Inflammatory cell infiltration, and a marked reduction in the lipid content of sebaceous glands. In the human trials, controlled cooling resulted in sebaceous gland damage and a diminished sebum level for two weeks, with minimal adverse effects on the surrounding tissue.

冷却过程导致皮脂腺细胞膜破裂，碱性磷酸酶活性降低，炎性细胞浸润以及皮脂腺脂质含量显着降低。在人体试验中，受控冷却导致皮脂腺损伤和皮脂水平降低两周，对周围组织的不良影响最小。

. It is widely acknowledged that thermal therapy and massage have therapeutic effects on meibomian glands. Hence, it is questioned whether cryogenic freezing can cause similar damage to the meibomian glands as it does to the skin, and whether such damage can simulate the pathophysiological process of atrophic meibomian gland dysfunction (MGD).

人们普遍认为热疗和按摩对睑板腺有治疗作用。因此，人们质疑低温冷冻是否会对睑板腺造成与皮肤相似的损伤，以及这种损伤是否可以模拟萎缩性睑板腺功能障碍（MGD）的病理生理过程。

What molecular mechanism changes can temperature or hypothermia induce in the meibomian glands? The following experiments were conducted to investigate these questions..

温度或体温过低会导致睑板腺发生什么分子机制的变化？进行了以下实验来研究这些问题。。

In this research, we introduce a murine model of meibomian gland dysfunction (MGD) that is induced by a targeted application of liquid nitrogen to the eyelid margin. This model is characterized by significant loss of meibomian gland acini and ductal structures, as well as decreasing secretion of meibomian glands.

在这项研究中，我们介绍了一种睑板腺功能障碍（MGD）的小鼠模型，该模型是通过将液氮靶向应用于眼睑边缘而诱导的。该模型的特征是睑板腺腺泡和导管结构的显着丧失，以及睑板腺的分泌减少。

No damage to other tissues. The development of this model is expected to advance our understanding of the pathophysiological alterations within meibomian glands subjected to cold-induced injury. Furthermore, it provides a platform for the exploration of novel therapeutic approaches for MGD and the potential for regenerative medicine strategies targeting the meibomian glands..

对其他组织无损伤。该模型的发展有望增进我们对遭受冷诱导损伤的睑板腺内病理生理改变的理解。此外，它为探索MGD的新型治疗方法以及针对睑板腺的再生医学策略的潜力提供了一个平台。。

Results

结果

Assessment of meibomian glands and other ocular surface tissues following liquid nitrogen injury to the eyelid margin

液氮损伤眼睑边缘后睑板腺和其他眼表组织的评估

Twenty-four hours after cryotherapy of the eyelid margin with liquid nitrogen, meibomian gland tissue from the mice was obtained for hematoxylin and eosin (H&E) staining and observation. Only the structure of the meibomian glands was found to be disrupted, with no changes observed in other structures.

液氮冷冻治疗眼睑边缘24小时后，取小鼠睑板腺组织进行苏木精-伊红（H＆E）染色和观察。发现只有睑板腺的结构被破坏，其他结构没有变化。

This indicates that the application of liquid nitrogen to the eyelid margin can specifically damage the acinar cells of the meibomian glands without affecting other tissue cells (as shown in Fig. .

这表明在眼睑边缘应用液氮可以特异性损伤睑板腺的腺泡细胞，而不会影响其他组织细胞（如图所示）。

Mice were evaluated at specific intervals of 7-, 14-, 21-, and 28-days post-application of liquid nitrogen cryotherapy to the eyelid margin. In comparison to the control group that received no treatment, there were observable changes in the eyelid margin’s smoothness and thickness, with a progressive increase in the visibility of corneal fluorescence over time (as depicted in Fig. .

在将液氮冷冻疗法应用于眼睑边缘后的7天，14天，21天和28天的特定间隔评估小鼠。与未接受治疗的对照组相比，眼睑边缘的平滑度和厚度有明显变化，随着时间的推移，角膜荧光的可见度逐渐增加（如图所示）。

A, B). The group observed at 21 days post-treatment demonstrated the most significant alterations, with the eyelid margin being the roughest and the corneal fluorescence staining being the most intense. The meibomian gland scoring criteria indicate pathological changes in the morphology of the eyelid margin following cryogenic injury.

A、 B）。治疗后21天观察到的组表现出最显着的改变，眼睑边缘最粗糙，角膜荧光染色最强烈。睑板腺评分标准表明低温损伤后眼睑边缘形态的病理变化。

(as depicted in Fig.

（如图所示）。

C).

C）。

The loss of meibomian glands is a significant diagnostic indicator of meibomian gland dysfunction (MGD)

睑板腺的丢失是睑板腺功能障碍（MGD）的重要诊断指标

. Subsequent to the application of liquid nitrogen cryotherapy to the eyelid margin, progressive morphological alterations of the meibomian glands were observed, reflecting glandular atrophy and the loss of principal ducts with the passage of time (as shown in Fig.

在将液氮冷冻疗法应用于眼睑边缘后，观察到睑板腺的进行性形态学改变，反映了腺体萎缩和随着时间的推移主管道的丧失（如图所示）。

Fig. 1

图1

Assessment of Meibomian acinar cells and Cornea within 24 h following liquid nitrogen Injury to the Eyelid Margin. (

液氮损伤眼睑边缘后24小时内评估睑板腺泡细胞和角膜。（笑声）(

) Representative H&E staining showing the eyelids of uninjured, 24 h post liquid nitrogen injury mouse. Panes indicate meibomian acinar cells, specifically ruptured meibomian acinar cells after liquid nitrogen treatment. Scale bars represent 50 μm and 20 μm (

)代表性的H＆E染色显示未受伤的液氮损伤后24小时小鼠的眼睑。窗格表示睑板腺泡细胞，特别是液氮处理后破裂的睑板腺泡细胞。比例尺代表50微米和20微米(

= 3). (

3.(

B类

) Representative H&E staining of corneas and conjunctiva show no significant damage and no increase of cell infiltration in the corneal stroma after the eyelid margin liquid nitrogen injury. Scale bars represent 20 μm (

)眼睑边缘液氮损伤后，角膜和结膜的代表性H＆E染色显示角膜基质中没有明显的损伤，细胞浸润也没有增加。比例尺代表20微米(

= 3).

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Fig. 2

图2

Assessment of MGs and cornea following liquid nitrogen injury of the eyelid margin. (

眼睑边缘液氮损伤后MGs和角膜的评估。（笑声）(

) Representative slit-lamp images showing the eyelids of uninjured mouse (Control) and those of day 7, day 14, day 21 and day 28 post liquid nitrogen injury mouse. The MG orifice plugging (black arrowheads), hypertrophic eyelid margin (green arrowheads) and telangiectasia (yellow arrowheads) are indicated.

)代表性裂隙灯图像显示未受伤小鼠（对照）和液氮损伤后第7天，第14天，第21天和第28天小鼠的眼睑。指示MG孔口堵塞（黑色箭头），肥厚的眼睑边缘（绿色箭头）和毛细血管扩张（黄色箭头）。

Scale bars represent 10 mm in ×16 images. (.

比例尺代表×16图像中的10毫米。（。

B类

) The fluorescein staining of the corneal surface indicates a healthy epithelium at days 7 post liquid nitrogen injury, with punctate damage seen at day 14, 21, 28 (red arrowheads). Scale bars represent 10 mm in ×16 images. (

)角膜表面的荧光素染色显示液氮损伤后第7天上皮健康，第14,21,28天可见点状损伤（红色箭头）。比例尺代表×16图像中的10毫米。（笑声）(

C级

) Meibomian Gland Scoring System

)睑板腺评分系统

. Data are shown as mean ± SD.

.数据显示为平均值±SD。

= 5, **

< 0.01, ****

< 0.0001.

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Fig. 3

图3

MG loss and morphological changes after liquid nitrogen. (

液氮后镁的损失和形态变化。（笑声）(

) Representative stereoscopic microscope images show MG loss and morphological changes of MGs after liquid nitrogen. The red rectangle represents the area of MG loss. Scale bars represent 1 mm (

)代表性的立体显微镜图像显示液氮后MG的损失和MG的形态变化。红色矩形表示MG损失的面积。比例尺代表1毫米(

= 3). (

3.(

B类

) The area of meibomian gland loss gradually increases after liquid nitrogen freezes the meibomian margin. Data are shown as mean ± SD.

)液氮冻结睑板边缘后，睑板腺损失面积逐渐增加。数据显示为平均值±SD。

= 3, **

< 0.01. , ****

< 0.0001.

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Changes of lipid synthesis after liquid nitrogen Injury to the eyelid margin

液氮损伤眼睑边缘后脂质合成的变化

Oil Red O staining indicates that after liquid nitrogen cryopreservation, the staining intensity of the meibomian gland acinar cells has become lighter. The morphology of the meibomian gland acini exhibited varying degrees of atrophy with extended observation time. The Oil Red O staining within the ducts shows incomplete filling (as shown in Fig. .

油红O染色表明，液氮冷冻保存后，睑板腺腺泡细胞的染色强度变轻。随着观察时间的延长，睑板腺腺泡的形态表现出不同程度的萎缩。导管内的油红O染色显示填充不完全（如图所示）。

A).

A）。

PPAR-γ (Peroxisome Proliferator-Activated Receptor gamma) plays a significant role in maintaining the morphology of Meibomian glands (MGs) and the differentiation of acinar cells, and it is also a critical factor in the lipid synthesis of MGs

. Immunofluorescent staining of PPAR-γ and western blot analysis has shown that following cryotherapy with liquid nitrogen applied to the eyelid margin, there was a decrease in PPAR-γ expression, with the 7-day and 21-day post-treatment groups exhibiting particularly significant reductions. Although there was an observed increase in PPAR-γ expression in the 14-day and 28-day post-treatment groups, the levels remained below the normal range.

PPAR-γ的免疫荧光染色和蛋白质印迹分析表明，在用液氮冷冻治疗眼睑边缘后，PPAR-γ表达降低，治疗后7天和21天组表现出特别显着的降低。尽管在治疗后14天和28天观察到PPAR-γ表达增加，但水平仍低于正常范围。

This suggests that the application of liquid nitrogen to the eyelid margin has led to a diminished capacity for lipid synthesis in the MGs and indicates an impairment in the differentiation of the acinar cells (as shown in Fig. .

这表明将液氮应用于眼睑边缘导致MGs中脂质合成能力降低，并表明腺泡细胞分化受损（如图所示）。

B–E). This suggests that cryogenic injury disrupts the structure of the cell membrane, thereby affecting the capacity for lipid synthesis.

B–E）。这表明低温损伤破坏了细胞膜的结构，从而影响了脂质合成的能力。

Fig. 4

图4

Histological changes and lipid synthesis of MGs after liquid nitrogen injury of the eyelid margin. (

液氮损伤眼睑边缘后MGs的组织学变化和脂质合成。（笑声）(

) Oil red O staining showing the meibomian gland ducts are partially filling. at days 7, 14, 21 and 28 post liquid nitrogen injury (circle). Some oil red O-labeled droplets indicated atrophy acini (black star). Scale bars represent 200 μm (

)油红O染色显示睑板腺导管部分充盈。在液氮损伤后第7,14,21和28天（圆圈）。一些油红O标记的液滴表明腺泡萎缩（黑星）。比例尺代表200微米(

= 3). (

3.(

B类

C级

) The expression of PPARγ in immunofluorescence significantly decreases on the 7th, 21st, and 28th days following liquid nitrogen cryopreservation. ****

)液氮冷冻保存后第7,21和28天，免疫荧光中PPARγ的表达显着降低****

< 0.0001. (

< 0.0001.(

) Western blot analysis shows the decline in PPARγ after liquid nitrogen treatment. The expression reduction was most significant at 7 days and 21days. **

)蛋白质印迹分析显示液氮处理后PPARγ下降。表达降低在7天和21天时最为显着**

< 0.01, ns > 0.05.

＜0.01，ns＞0.05。

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Inflammation of MGs after liquid nitrogen injury of the eyelid margin

液氮损伤眼睑边缘后MGs的炎症

Inflammatory infiltration is a characteristic feature of Meibomian Gland Dysfunction (MGD)

炎症浸润是睑板腺功能障碍（MGD）的特征

. The expression levels of IL-1β and IL-18, as well as the immunofluorescence observation of TNFα, revealed that inflammatory cytokines peaked on the 7th day, likely due to the acute phase of injury. However, as the observation period of the experiment extended, inflammation did not show a significant decrease but rather remained at a higher level.

IL-1β和IL-18的表达水平以及TNFα的免疫荧光观察显示，炎性细胞因子在第7天达到峰值，可能是由于损伤的急性期。然而，随着实验观察期的延长，炎症没有显示出显着的下降，而是保持在更高的水平。

Observations on the 21st and 28th days indicated that the inflammatory cytokines were still higher than those in the control group. We infer that this may be the reason for the further decline and deterioration of meibomian gland function (as shown in Fig. .

第21天和第28天的观察表明，炎性细胞因子仍高于对照组。我们推断这可能是睑板腺功能进一步下降和恶化的原因（如图所示）。

A, B, D).

A、 B，D）。

Fig. 5

图5

Inflammation of MGs after liquid nitrogen injury of eyelid margin. (

液氮损伤眼睑边缘后MGs的炎症。（笑声）(

) Gene expression levels of IL-1β, IL-18 were upregulated in the MG of mice post liquid nitrogen injury. ****

)液氮损伤后小鼠MG中IL-1β，IL-18的基因表达水平上调****

< 0.0001, ***

< 0.001. (

< 0.001.(

B类

) Immunofluorescence staining of TNFα was upregulated in the MG post liquid nitrogen injury. Scale bars represent 50 μm (

)液氮损伤后MG中TNFα的免疫荧光染色上调。比例尺代表50微米(

= 3). ****

< 0.0001. (

< 0.0001.(

C级

) Immunofluorescence staining of K14 was downregulated in the

)K14的免疫荧光染色在

meibomian gland acinar cells

睑板腺腺泡细胞

post liquid nitrogen injury. Scale bars represent 50 μm (

液氮损伤后。比例尺代表50微米(

= 3). ****

< 0.0001.

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We performed CD68 + iNOS double-labeling fluorescence staining to assess inflammatory cell infiltration in the meibomian glands of different groups. Observations revealed that CD68-positive cells were present at all stages following liquid nitrogen-induced eyelid margin injury, with iNOS-positive cells being more prominent in the early stages but decreasing at later stages (as shown in supplementary Figure)..

。观察显示，液氮诱导的眼睑边缘损伤后，CD68阳性细胞在所有阶段都存在，iNOS阳性细胞在早期阶段更为突出，但在后期减少（如补充图所示）。。

At day 7, CD68(+) cells were predominantly located at the apex of the acini, with greater accumulation around the acini than at the eyelid margin. iNOS(+) cells were more abundant near the acini and fewer at the eyelid margin. By day 14, CD68(+) cells remained near the acini with minimal clustering at the eyelid margin, while iNOS(+) cells had significantly decreased compared to the day 7 group and were almost absent at the eyelid margin.

在第7天，CD68（+）细胞主要位于腺泡的顶端，腺泡周围的积累大于眼睑边缘。iNOS（+）细胞在腺泡附近更丰富，在眼睑边缘更少。到第14天，CD68（+）细胞保持在腺泡附近，在眼睑边缘聚集最少，而iNOS（+）细胞与第7天组相比显着减少，并且在眼睑边缘几乎不存在。

At days 21 and 28, a small number of CD68(+) cells were observed near the acini, with no significant changes in their presence at the eyelid margin, and iNOS(+) cells were scarcely detected in either location. These changes are consistent with the pathological alterations reported in dermatology, where pro-inflammatory macrophages dominate in the early stages of cryotherapy-induced skin injury, and repair-oriented, anti-inflammatory cells become predominant at later stages.

在第21天和第28天，在腺泡附近观察到少量CD68（+）细胞，其在眼睑边缘的存在没有显着变化，并且在任一位置几乎都没有检测到iNOS（+）细胞。这些变化与皮肤病学报道的病理改变一致，其中促炎巨噬细胞在冷冻疗法诱导的皮肤损伤的早期阶段占主导地位，并且以修复为导向的抗炎细胞在后期占主导地位。

Cell proliferation and apoptosis in MGs after liquid nitrogen Injury to the eyelid margin

液氮损伤眼睑边缘后MGs细胞增殖和凋亡

Ki67 serves as a marker for cells that are actively proliferating, including those within the meibomian glands and various other tissues

Ki67是活跃增殖细胞的标志物，包括睑板腺和其他各种组织内的细胞

. Under normal physiological conditions, Ki67 is expressed in the basal and ductal cells of the meibomian gland acini, indicating cell proliferation. In this model, a decrease in Ki67 expression was observed. At 14 days post-treatment, expression was confined to partial basal areas of the acini. By day 21, Ki67 expression was significantly reduced, and minimal expression was detected at the 28-day assessment.

在正常生理条件下，Ki67在睑板腺腺泡的基底细胞和导管细胞中表达，表明细胞增殖。在该模型中，观察到Ki67表达降低。治疗后14天，表达仅限于腺泡的部分基底区域。到第21天，Ki67表达显着降低，并且在28天评估时检测到最小表达。

This continuous decrease in Ki67 expression may reflect the acinar cells’ response to cold-induced injury, indicating a sustained decline in cell proliferative function within the meibomian glands and maintenance at a low level. The reduction in Ki67-positive cells may also suggest the presence of meibomian gland dysfunction and/or impaired regenerative capacity of the acinar cells (as shown in Fig. .

Ki67表达的持续下降可能反映了腺泡细胞对冷诱导损伤的反应，表明睑板腺内细胞增殖功能持续下降，并维持在低水平。Ki67阳性细胞的减少也可能表明存在睑板腺功能障碍和/或腺泡细胞的再生能力受损（如图所示）。

A, B).

A、 B）。

Fig. 6

图6

Cell proliferation and apoptosis in MGs after liquid nitrogen injury. (

液氮损伤后MGs中的细胞增殖和凋亡。（笑声）(

B类

) Immunofluorescence staining of Ki67 and positive cell counting indicates a decreased in positive cells after liquid nitrogen injury, with labelled cells rarely detected at day 28. Scale bars represent 50 μm (

)Ki67的免疫荧光染色和阳性细胞计数表明液氮损伤后阳性细胞减少，在第28天很少检测到标记细胞。比例尺代表50微米(

= 3). Data are shown as mean ± SD (

数据显示为平均SD(

= 3), ****

< 0.0001. (

< 0.0001.(

C级

) TUNEL staining and positive cell counting shows abundant positive cells around MGs post liquid nitrogen injury Scale bars represent 50 μm (

)TUNEL染色和阳性细胞计数显示液氮损伤后MGs周围有大量阳性细胞，比例尺为50米(

= 3). ****

< 0.0001.

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The TUNEL staining indicates that apoptosis is consistently present following liquid nitrogen injury, showing an upward trend from the 7th to the 14th day of observation. From day 14 to day 28, apoptosis can be essentially maintained at a relatively high level (as shown in Fig.

TUNEL染色表明液氮损伤后细胞凋亡持续存在，从观察的第7天到第14天呈上升趋势。从第14天到第28天，细胞凋亡基本上可以维持在相对较高的水平（如图所示）。

C, D).

C、 D）。

Basal epithelial cells displayed K14 staining, indicating the presence of intermediate filaments in the epithelial cell skeleton, which is one of the markers for basal cells

基底上皮细胞显示K14染色，表明上皮细胞骨架中存在中间丝，这是基底细胞的标志之一

. Our data reveal a decrease in K14 staining following localized cryogenic injury, suggesting atrophy of the meibomian gland acini (as shown in Fig.

我们的数据显示局部低温损伤后K14染色减少，提示睑板腺腺泡萎缩（如图所示）。

C, E).

C、 E）。

Keratinization of MG after liquid nitrogen Injury to the eyelid margin

液氮损伤眼睑边缘后MG的角化

Hyper keratinization is a definitive pathological sign of obstructive Meibomian Gland Dysfunction (MGD)

过度角化是阻塞性睑板腺功能障碍（MGD）的明确病理征象

, which can lead to ductal dilation and acinar atrophy. K10 (Keratin 10) is a biomarker for terminally differentiated keratinized epithelium, and its expression is relatively low in the central ducts of normal meibomian glands

，这可能导致导管扩张和腺泡萎缩。K10（角蛋白10）是终末分化角化上皮的生物标志物，其在正常睑板腺中央导管中的表达相对较低

. Beginning on the seventh day after cryotherapy of the eyelid margin with liquid nitrogen, immunofluorescence revealed the emergence of K10 expression in the acini, and an increase in K10 expression in the ducts with the extension of the observation period. Additionally, areas of meibomian gland atrophy and loss exhibited high levels of K10 expression.

从液氮冷冻治疗眼睑边缘后的第七天开始，免疫荧光显示腺泡中出现K10表达，随着观察期的延长，导管中K10表达增加。此外，睑板腺萎缩和丢失的区域表现出高水平的K10表达。

This indicates a significant increase in keratinization following the application of liquid nitrogen to the eyelid margin, and that this hyper-keratinized state persists over time (as shown in Fig. .

这表明在将液氮应用于眼睑边缘后角化显着增加，并且这种过度角化状态随着时间的推移而持续（如图所示）。

A).

A）。

Sprr1b (Small Proline-Rich Protein 1B) is recognized as one of the markers for keratinization

Sprr1b（富含脯氨酸的小蛋白1B）被认为是角化的标志物之一

. To further validate the occurrence of keratinization, we assessed the mRNA expression levels of Sprr1b and observed an increase in its expression following treatment with liquid nitrogen. Notably, the 7-day and 30-day post-treatment groups exhibited particularly significant elevations in Sprr1b expression.

为了进一步验证角化的发生，我们评估了Sprr1b的mRNA表达水平，并观察到液氮处理后其表达增加。值得注意的是，治疗后7天和30天的组在Sprr1b表达方面表现出特别显着的升高。

Although there was a slight decrease in the 14-day and 21-day groups, the levels remained higher than those of the control group (as shown in Fig. .

尽管14天和21天组略有下降，但水平仍高于对照组（如图所示）。

B).

B）。

Fig. 7

图7

keratinization of MGs after liquid nitrogen injury of eyelid margin. (

液氮损伤眼睑边缘后MGs的角化。（笑声）(

) Immunofluorescence staining of K10 revealing hyper-keratinization and thickening of the MG duct at days 21 and 28 post liquid nitrogen injury. Scale bars represent 50 μm (

)K10的免疫荧光染色显示液氮损伤后第21天和第28天MG管过度角化和增厚。比例尺代表50微米(

= 3). (

3.(

B类

) Gene expression levels of Sprr1β was upregulated in the MG of mice post liquid nitrogen injury. ****

)****

< 0.001, *

< 0.05.

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Discussion

讨论

We have delineated a mouse model of atrophic meibomian Gland Dysfunction (MGD). In our study, we found that liquid nitrogen can specifically disrupt the cell membranes of acinar cells in the meibomian glands in the acute post-injury phase (within 24 h), without damaging adjacent tissues such as the orbicularis oculi muscle or the cornea.

我们已经描述了萎缩性睑板腺功能障碍（MGD）的小鼠模型。在我们的研究中，我们发现液氮可以在急性损伤后阶段（24小时内）特异性破坏睑板腺腺泡细胞的细胞膜，而不会损伤邻近组织，如眼轮匝肌或角膜。

And then, there is a significant decrease in lipid content following injury. Concurrently, the meibomian glands have shown varying degrees of atrophy and loss, the eyelid margins have become thickened and rounded, and there is neovascularization. Commencing on day 14, the cornea exhibited significant fluorescence staining..

然后，损伤后脂质含量显着降低。同时，睑板腺显示出不同程度的萎缩和丧失，眼睑边缘变厚和变圆，并且有新血管形成。从第14天开始，角膜表现出明显的荧光染色。。

Since the use of the liquid nitrogen freezing method to create a meibomian gland dysfunction (MGD) model is being applied for the first time, there are no ophthalmology-specific references to explain the underlying mechanisms. The inspiration for this model comes from dermatology, where the mechanisms of similar models have been better studied.

。这个模型的灵感来自皮肤病学，类似模型的机制已经得到了更好的研究。

Based on the existing literature and the current experimental results, the possible mechanisms can be summarized as follows:.

根据现有文献和当前的实验结果，可能的机制可以总结如下：。

First, local freezing leads to a decrease in the number of acinar cells capable of producing meibum. HE staining at 24 h reveals significant structural damage to acinar cells, with disrupted cell membranes and compromised cellular integrity. Cryotherapy selectively damages lipid-rich cells while minimizing injury to surrounding tissues.

首先，局部冷冻导致能够产生睑板的腺泡细胞数量减少。24小时的HE染色显示腺泡细胞受到明显的结构损伤，细胞膜被破坏，细胞完整性受损。冷冻疗法选择性地破坏富含脂质的细胞，同时最大限度地减少对周围组织的损伤。

. Intracellular lipids crystallize at temperatures higher than the freezing point of tissue fluid

细胞内脂质在高于组织液冰点的温度下结晶

. Additionally, freezing disrupts cell membranes, reducing the number of functional acinar cells and their ability to produce lipids. The initiating factor in this model is acinar damage, differing from other MGD models where atrophy occurs secondary to mechanical obstruction. Dermatological studies have reported that cryotherapy affects the differentiation process of basal stem cells in the skin, which might explain why acinar cells in this model fail to regenerate in a timely manner.

此外，冷冻会破坏细胞膜，减少功能性腺泡细胞的数量及其产生脂质的能力。该模型的起始因素是腺泡损伤，与其他MGD模型不同，后者继发于机械性梗阻而发生萎缩。皮肤病学研究报道，冷冻疗法会影响皮肤中基底干细胞的分化过程，这可能解释了为什么该模型中的腺泡细胞无法及时再生。

Second, following acinar damage, a series of inflammatory responses occur within the meibomian gland. At different time points after injury, inflammatory cell infiltration is observed, primarily around the acinar tissue, along with the accumulation of inflammatory mediators. This process resembles changes seen in subcutaneous fat tissue after cryotherapy.

其次，腺泡损伤后，睑板腺内发生一系列炎症反应。在损伤后的不同时间点，观察到炎性细胞浸润，主要在腺泡组织周围，以及炎性介质的积累。这个过程类似于冷冻治疗后皮下脂肪组织的变化。

Dieter Manstein’s study demonstrated increasing infiltration of inflammatory cells and progressive destruction of lipid-rich cells up to 30 days after cryotherapy in porcine skin (2). Similarly, Mathew M observed comparable results in human skin following cryotherapy, paralleling the changes observed in this model.

。类似地，Mathew M在冷冻疗法后在人体皮肤中观察到可比的结果，与在该模型中观察到的变化平行。

Third, reduced meibum secretion destabilizes the tear film and increases tear osmolarity, triggering ocular surface inflammation and forming a vicious cycle. This inflammation may impair the repair of meibomian gland tissue and contribute to keratinization of the gland, likely due to the persistent accumulation of inflammatory mediators.

第三，睑板分泌减少会破坏泪膜的稳定性并增加泪液渗透压，引发眼表炎症并形成恶性循环。这种炎症可能会损害睑板腺组织的修复，并导致腺体角化，这可能是由于炎症介质的持续积累。

Lastly, freezing may lead to ischemia in meibomian gland tissue. By day 7 of the model, neovascularization was observed at the eyelid margin, possibly as a compensatory response to cryotherapy-induced ischemia. Freeze injury can cause indirect damage,

最后，冷冻可能导致睑板腺组织缺血。到模型的第7天，在眼睑边缘观察到新血管形成，可能是对冷冻疗法诱导的缺血的补偿反应。冻伤会造成间接伤害，

such as vasoconstriction

例如血管收缩

, endothelial damage, and thrombosis, leading to localized ischemia. However, experimental evidence supporting this mechanism is insufficient and requires further investigation

，内皮损伤和血栓形成，导致局部缺血。然而，支持这种机制的实验证据不足，需要进一步研究

Our findings are in accord with recent studies indicating that these observations are in accordance with the clinical presentation of atrophic Meibomian Gland Dysfunction (MGD)

我们的发现与最近的研究一致，表明这些观察结果与萎缩性睑板腺功能障碍（MGD）的临床表现一致

. The clinical manifestations of age-related MGD mice were also very similar to those of this model

与年龄相关的MGD小鼠的临床表现也与该模型非常相似

. However, Knockout-targeted genes are often key to lipid metabolism or ectoderm development, such as SCD1-null (SCD1 −/−) mice, K5-GR mice, Krox20-cKO mice, and Tabby mice

然而，敲除靶向基因通常是脂质代谢或外胚层发育的关键，例如SCD1-null（SCD1-/-）小鼠，K5-GR小鼠，Krox20 cKO小鼠和Tabby小鼠

. These models often exhibit dysplasia of the meibomian glands, a gene knockout mouse that not only alters the function of the meibomian glands, but also causes significant morphological changes in the meibomian glands. Although these gene knockout models can explain the effects of the gene on the development and function of meibomian glands at the molecular or pathway level from some perspectives, most of them cannot mimic the pathophysiological process of patients with meibomian gland dysfunction in clinical practice, because the vast majority of patients have meibomian gland dysplasia or even abnormal, and the changes in meibomian gland morphology and function are also caused by various acquired factors, and these gene knockout models do not explain how acquired factors affect the gene.

这些模型通常表现出睑板腺发育异常，这是一种基因敲除小鼠，不仅改变了睑板腺的功能，而且还引起了睑板腺的显着形态变化。虽然这些基因敲除模型可以从某些角度解释该基因在分子或途径水平上对睑板腺发育和功能的影响，但大多数基因敲除模型不能模拟临床实践中睑板腺功能障碍患者的病理生理过程，因为绝大多数患者患有睑板腺发育异常甚至异常，睑板腺形态和功能的变化也是由各种后天因素引起的，这些基因敲除模型不能解释后天因素如何影响该基因。

Therefore, there are still limitations to the study of pathogenic mechanisms. At the same time, gene knockout mice are the knockout of whole body cells, and it is impossible to specifically knock out one or several genes of meibomian gland tissue, which will inevitably cause damage to the ocular surface and other tissues and organs of the body to varying degrees, such as corneal neovascularization, corneal epithelial keratosis, and even kidney function damage in mice, etc., these side injuries are not desirable to us, and are not conducive to observing the pathophysiological changes that should occur on the ocular surface under MGD.

因此，致病机制的研究仍然存在局限性。同时，基因敲除小鼠是全身细胞的敲除，不可能特异性敲除睑板腺组织的一个或几个基因，这将不可避免地对眼表和身体其他组织器官造成不同程度的损伤，如角膜新生血管，角膜上皮角化病，甚至小鼠肾功能损伤等，这些侧面损伤对我们来说是不可取的，也不利于观察MGD下眼表应发生的病理生理变化。

In addition, genetic selection in knockout mice is also limited. I have to admit that the knockout genes that exist at present do have an impact on meibomian gland function, but there is no definite conclusion that these genes must be key and necessary influencing factors, and a large part of the genes that affect meibomian gland function may not be able t.

此外，基因敲除小鼠的遗传选择也受到限制。我必须承认，目前存在的敲除基因确实对睑板腺功能有影响，但没有明确的结论表明这些基因一定是关键和必要的影响因素，而影响睑板腺功能的大部分基因可能无法。

The reduction in the number of meibomian gland acinar cells leads to decreased expression of PPARγ, which may also be the initiating cause of atrophic MGD. PPARγ has anti-inflammatory effects, and the expression of inflammatory factors can be regulated through the PPAR-γ signaling pathway itself, and its role is to inhibit the expression of inflammatory factors, such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), etc., and promote the production of anti-inflammatory cytokines.

。PPARγ具有抗炎作用，炎症因子的表达可以通过PPAR-γ信号通路本身进行调节，其作用是抑制肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）等炎症因子的表达，促进抗炎细胞因子的产生。

. Observations indicate that IL-1β, IL-18, and TNFα in this model accumulate and increase over time, particularly noticeable by the 21st and 28th days, which we believe contributes to the further deterioration of acinar cell function. The apoptosis of acinar cells becomes increasingly evident, and the expression of the basal cell marker K14 also gradually decreases.

观察结果表明，该模型中的IL-1β，IL-18和TNFα随时间积累并增加，在第21天和第28天尤其明显，我们认为这有助于腺泡细胞功能的进一步恶化。腺泡细胞的凋亡变得越来越明显，基底细胞标志物K14的表达也逐渐降低。

This suggests that in the advanced stages of atrophic MGD, the self-renewal capacity of the acini has declined, possibly due to issues with the multipotency of basal cells. However, the specific mechanisms still require further experimental research to be revealed, which will also be the direction of future research for our group..

这表明在萎缩性MGD的晚期，腺泡的自我更新能力下降，可能是由于基底细胞的多能性问题。然而，具体的机制仍需要进一步的实验研究来揭示，这也将是我们小组未来研究的方向。。

Surprisingly, upon extending the observation period in this model, it was found that around 60 days post cryotherapy with liquid nitrogen, a majority of the model mice exhibited regenerative capabilities of the meibomian glands, with the post-repair conditions largely resembling those prior to treatment.

令人惊讶的是，在延长该模型的观察期后，发现液氮冷冻治疗后约60天，大多数模型小鼠表现出睑板腺的再生能力，修复后的条件与治疗前基本相似。

The localization of meibomian gland stem/progenitor cells is a current focal point and a matter of debate in research, with the majority of studies concentrating on animal models of embryonic development.

睑板腺干/祖细胞的定位是目前研究的焦点和争论的问题，大多数研究集中在胚胎发育的动物模型上。

. However, these models do not elucidate the mechanisms and processes by which adult meibomian glands self-renew. This indicates that the meibomian glands indeed possess regenerative potential, and this model can also serve as an animal model for subsequent studies tracking the localization of meibomian gland stem/progenitor cells, the pathways of acinar regeneration, and the mechanisms underlying acinar regeneration..

然而，这些模型并没有阐明成年睑板腺自我更新的机制和过程。这表明睑板腺确实具有再生潜力，该模型也可以作为后续研究的动物模型，用于追踪睑板腺干/祖细胞的定位，腺泡再生途径以及腺泡再生的机制。。

Materials and methods

材料和方法

Materials

Rabbit anti-Cytokeratin 10 (K10, ab76318), anti-Cytokeratin 14 (K14, ab181595), anti-Ki67 (ab16667), anti-PPAR-γ (ab45036). Alexa Fluor 488-conjugated IgG (A11055, A21206) and Alexa Fluor 594conjugated IgG (A11058) were from Invitrogen (Eugene, OR, USA). 40,6-diamidino- 2-phenylindole (DAPI; H-1200)..

兔抗细胞角蛋白10（K10，ab76318），抗细胞角蛋白14（K14，ab181595），抗Ki67（ab16667），抗PPAR-γ（ab45036）。Alexa Fluor 488偶联的IgG（A11055，A21206）和Alexa Fluor 594偶联的IgG（A11058）来自Invitrogen（美国俄勒冈州尤金）。40,6-二脒基-2-苯基吲哚（DAPI；H-1200）。。

Animals

动物

Eight-week-old male C57/BL6 mouses were from Changchun-bio (China),

八周大的雄性C57/BL6小鼠来自长春生物（中国），

= 80. Experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research, and with approval of the Animal Ethical Committee of Harbin medical University. Animals were given free access to standard rodent chow and water and kept in a standard pathogen-free environment at 25 °C ± 1 °C, relative humidity 60%±10%, and alternating 12 h light-dark cycles (from 8:00 AM to 8:00 PM)..

根据视觉与眼科研究协会（ARVO）关于在眼科和视觉研究中使用动物的声明，并经哈尔滨医科大学动物伦理委员会批准，进行了实验程序。让动物自由获取标准啮齿动物食物和水，并将其保存在25°C±1°C，相对湿度60%±10%的标准无病原体环境中，并交替进行12小时的明暗循环（从上午8:00到晚上8:00）。。

Animal treatment

动物治疗

In this study, we administered liquid nitrogen cryotherapy to the eyelid margins of mouse in the treatment group. This procedure was carried out under general anesthesia induced by inhalation of 4% isoflurane (Sinopharm Chemical Reagent Co., Ltd., China) in oxygen, followed by topical application of 0.4% oxybuprocaine hydrochloride eye drops (Santen Pharmaceutical, Japan) for local anesthesia of the eyelid margins.

在这项研究中，我们对治疗组小鼠的眼睑边缘进行了液氮冷冻治疗。该手术是在氧气中吸入4%异氟烷（国药集团化学试剂有限公司，中国）诱导的全身麻醉下进行的，然后局部应用0.4%盐酸氧布卡因滴眼液（日本Santen Pharmaceutical）进行眼睑边缘的局部麻醉。

Liquid nitrogen was placed in a thermos flask and then the temperature was conducted using a specially crafted pure copper device to apply cryotherapy to both the upper and lower eyelid margins. The temperature of the device that touches the eyelids can be maintained at minus 110 degrees Celsius..

将液氮置于热水瓶中，然后使用特制的纯铜装置进行温度测量，以对上眼睑和下眼睑边缘进行冷冻治疗。接触眼睑的设备的温度可以保持在零下110摄氏度。。

degrees or so. The central two thirds of the upper eyelid margin of the selected eye was treated by applying the aforementioned device for 30 s (as shown in Fig.

度左右。通过应用上述装置30秒来治疗所选眼睛上眼睑边缘的中央三分之二（如图所示）。

). In the control group, mice received no treatment and were housed in the same environment as those that underwent liquid nitrogen cryotherapy. Mice in the experimental group were sacrificed at 7, 14, 21, and 28 days following the eyelid margin cryotherapy. The control group mice were sacrificed after 28 days.

)。在对照组中，小鼠未接受治疗，并被安置在与接受液氮冷冻治疗的小鼠相同的环境中。实验组小鼠在眼睑边缘冷冻治疗后7,14,21和28天处死。对照组小鼠在28天后处死。

At the end of the experiment, all animals were euthanized with a lethal dose of sodium pentobarbital (80 mg/kg BW), followed by cervical dislocation to ensure death..

在实验结束时，所有动物都被致命剂量的戊巴比妥钠（80 mg/kg体重）安乐死，然后颈椎脱位以确保死亡。。

Fig. 8

图8

Schematic illustration of the procedure to perform local Liquid nitrogen cryotherapy on mouse MG orifices. (

对小鼠MG孔进行局部液氮冷冻治疗的程序的示意图。（笑声）(

) Refrigeration units. (

)制冷装置。（笑声）(

B类

) About 13 min after filling with liquid nitrogen, the temperature of the unit tends to stabilize at -100 degrees Celsius ± 15 degrees Celsius. (

)充入液氮约13分钟后，装置的温度趋于稳定在-100摄氏度±15摄氏度。（笑声）(

C级

) Flow diagram.

)流程图。

Full size image

全尺寸图像

Slit-lamp microscopy examination

裂隙灯显微镜检查

After the induction of general anesthesia, the patency of the meibomian gland (MG) orifices was evaluated utilizing a slit-lamp microscope. The state of the orifices was documented with a digital camera, and the quantity of obstructed orifices out of a total of ten MG orifices located centrally in the upper eyelid was enumerated.

全身麻醉诱导后，使用裂隙灯显微镜评估睑板腺（MG）孔的通畅性。用数码相机记录了孔的状态，并列举了位于上眼睑中央的总共十个MG孔中阻塞的孔的数量。

Orifices were categorized as obstructed if they exhibited opacity and swelling. To appraise the integrity of the corneal epithelium, one microliter of a 1% solution of liquid sodium fluorescein (Jingmingxin Co., Ltd., Tianjin, China) was instilled into the conjunctival sac. The corneal epithelium’s uptake of fluorescein was subsequently observed after a 90-second interval, employing a slit-lamp microscope equipped with a cobalt blue filter.

如果孔表现出不透明和肿胀，则将其归类为阻塞。。随后，使用配备有钴蓝滤光片的裂隙灯显微镜，在90秒的间隔后观察角膜上皮对荧光素的摄取。

A consistent ophthalmologist captured all images using an identical camera and settings throughout the examination process..

在整个检查过程中，一致的眼科医生使用相同的相机和设置拍摄了所有图像。。

Histology

组织学

Post-injury collection of mouse eyelids was conducted at specific time points: 0, 1, 7, 14, 21, and 28 days. These eyelid samples were then embedded either in Optimal Cutting Temperature (OCT) medium or paraffin. From these blocks, sagittal sections of the tissue, measuring 6 micrometers in thickness, were prepared with three sections per slide, and three slides were created per animal, resulting in a total of 3 animals per experimental group.

在特定时间点进行小鼠眼睑的损伤后收集：0,1,7,14,21和28天。然后将这些眼睑样品包埋在最佳切割温度（OCT）培养基或石蜡中。从这些块中，制备厚度为6微米的组织矢状切片，每个载玻片有三个切片，每只动物创建三个载玻片，每个实验组总共有3只动物。

Hematoxylin and eosin (H&E) staining was applied to the paraffin-embedded sections to assess tissue morphology. Meanwhile, immunofluorescent staining and Oil Red O staining were conducted on the OCT-embedded frozen sections to further analyze cellular and lipid characteristics, respectively..

将苏木精和曙红（H＆E）染色应用于石蜡包埋的切片以评估组织形态。同时，在OCT包埋的冷冻切片上进行免疫荧光染色和油红O染色，分别进一步分析细胞和脂质特征。。

Oil red O staining

油红O染色

The accumulation of lipids in the meibomian glands (MGs) was determined by evaluating the intensity of the Oil Red O stain. The frozen sections of the eyelids were initially fixed in a 5% paraformaldehyde solution for a duration of 10 min, followed by a wash in phosphate-buffered saline (PBS) for 5 min.

通过评估油红O染色的强度来确定睑板腺（MG）中脂质的积累。首先将眼睑的冷冻切片在5%多聚甲醛溶液中固定10分钟，然后在磷酸盐缓冲盐水（PBS）中洗涤5分钟。

Subsequently, the sections were stained with a freshly prepared Oil Red O solution for 25 min. After staining, the sections were rinsed with PBS for another 5 min. Finally, the sections were mounted using a 90% glycerol solution and examined under a light microscope to visualize the lipid deposits..

。染色后，将切片用PBS再冲洗5分钟。最后，使用90%甘油溶液固定切片，并在光学显微镜下检查以观察脂质沉积物。。

Immunofluorescent staining

免疫荧光染色

For the immunofluorescent staining procedure, cryosections of mouse eyelids, 6 micrometers in thickness, were treated by being fixed in cold acetone at -20 degrees Celsius for 10 min. This was followed by a series of three washes with phosphate-buffered saline (PBS), each lasting for 5 min. The sections were then permeabilized with a 0.3% Triton X-100 solution for 30 min and rinsed again with PBS in triplicate, with each wash being 5 min long..

对于免疫荧光染色程序，将厚度为6微米的小鼠眼睑冷冻切片在-20℃的冷丙酮中固定10分钟。然后用磷酸盐缓冲盐水（PBS）洗涤三次，每次持续5分钟。然后将切片用0.3%Triton X-100溶液透化30分钟，并再次用PBS一式三份冲洗，每次冲洗5分钟。。

Subsequently, the sections were blocked to reduce non-specific binding by incubating them with 2% bovine serum albumin (BSA) in PBS for 60 min at ambient temperature. The primary antibodies, K14, K10 (diluted 1:500) and Ki67, PPARγ (diluted 1:1000), were applied to the sections, which were then incubated for 16 h at 4 degrees Celsius.

随后，通过将切片与PBS中的2%牛血清白蛋白（BSA）在环境温度下孵育60分钟，封闭切片以减少非特异性结合。将一抗K14，K10（1:500稀释）和Ki67，PPARγ（1:1000稀释）应用于切片，然后在4℃下孵育16小时。

To ensure specificity, negative control sections were treated with a non-relevant isotype control antibody in place of the primary antibody..

为了确保特异性，用不相关的同种型对照抗体代替一抗处理阴性对照切片。。

Following the incubation with primary antibodies, the sections were washed extensively with PBS for 10 min per wash, three times in total. The sections were then incubated with the secondary antibodies, either Alexa Fluor 488-conjugated IgG (diluted 1:250) or Alexa Fluor 594-conjugated IgG (diluted 1:250), for 1 h at room temperature in a dark environment to protect the fluorescent labels from light-induced bleaching..

与一抗孵育后，每次用PBS充分洗涤切片10分钟，总共三次。然后将切片与二抗（Alexa Fluor 488缀合的IgG（1:250稀释）或Alexa Fluor 594缀合的IgG（1:250稀释））在室温下在黑暗环境中孵育1小时，以保护荧光标记免受光诱导的漂白。。

Finally, the sections were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei and then examined using a fluorescence microscope to capture images of the stained cells.

最后，将切片用4'，6-二脒基-2-苯基吲哚（DAPI）复染以显现细胞核，然后使用荧光显微镜检查以捕获染色细胞的图像。

TUNEL assay

标记法

To assess cellular apoptosis, we employed the TUNEL assay kit from Elabscience One-step TUNEL In Situ Apoptosis Kit (E-CK-A320). Initially, paraffin-embedded tissue sections underwent rehydration and were then treated with a Proteinase-K solution in Tris/HCL buffer at a pH of 7.4, with a concentration of 10 mM, for a duration of 30 min at a controlled temperature of 37 °C.

为了评估细胞凋亡，我们使用了Elabscience One step TUNEL原位凋亡试剂盒（E-CK-A320）的TUNEL检测试剂盒。最初，对石蜡包埋的组织切片进行再水化，然后在37°C的受控温度下，用Tris/HCL缓冲液中的蛋白酶K溶液（pH为7.4，浓度为10 mM）处理30分钟。

Subsequently, the sections were thoroughly rinsed with Phosphate-Buffered Saline (PBS), repeating the process three times for 5 min each to ensure complete removal of the Proteinase-K. Following the washes, 50 µl of the TUNEL reaction cocktail was applied to the sections, which were then incubated in the dark at 37 °C for a period of 1 h.

。

Post-incubation, the sections were again subjected to three rounds of washing with PBS for 5 min each to eliminate any unbound reaction components. To visualize the nuclei, the sections were subsequently counterstained with 4’,6-diamidino-2-phenylindole (DAPI), a fluorescent stain that binds specifically to DNA.

孵育后，将切片再次用PBS洗涤三轮，每次5分钟，以消除任何未结合的反应组分。为了可视化细胞核，随后将切片用4'，6-二脒基-2-苯基吲哚（DAPI）复染，DAPI是一种与DNA特异性结合的荧光染色剂。

After the counterstaining procedure, the sections were mounted onto slides for preservation and clarity. Finally, the stained sections were examined and documented using a high-resolution microscope (model DM2500 from Leica Microsystems), capturing images that reveal the presence of apoptotic cells through the TUNEL assay..

复染程序后，将切片安装在载玻片上以保存和清晰。最后，使用高分辨率显微镜（Leica Microsystems的DM2500型）检查并记录染色切片，通过TUNEL分析捕获显示凋亡细胞存在的图像。。

Western blot analysis

蛋白质印迹分析

Isolated meibomian gland (MG) samples were collected using a cold lysis buffer supplemented with a cocktail of protease and phosphatase inhibitors (Catalog No. 78440, Thermo Fisher Scientific). Protein content was determined utilizing a BCA Protein Assay Kit (Catalog No. 23225, Thermo Fisher Scientific).

使用补充有蛋白酶和磷酸酶抑制剂混合物的冷裂解缓冲液（目录号78440，Thermo Fisher Scientific）收集分离的睑板腺（MG）样品。使用BCA蛋白质测定试剂盒（目录号23225，Thermo Fisher Scientific）测定蛋白质含量。

Each experimental set comprised three replicates, with each replicate being a composite of MGs harvested from both ocular glands of an individual mouse. A standardized quantity of protein lysate (20 µg) from each sample was loaded and resolved on 10% tricine gels, followed by transfer to a polyvinylidene fluoride (PVDF) membrane.

每个实验组包括三个重复，每个重复是从单个小鼠的两个眼腺收获的MG的复合物。将来自每个样品的标准量的蛋白质裂解物（20µg）上样并在10%tricine凝胶上分离，然后转移到聚偏二氟乙烯（PVDF）膜上。

The membranes were then blocked with 5% bovine serum albumin (BSA) for 60 min. Incubated with primary antibodies specific for PPAR-γ (dilution 1:1000) or β-actin (dilution 1:8000) after cropping at 4 °C overnight. After three rinses with Tris-buffered saline with 0.05% Tween 20 for 10-minute intervals, the membranes were treated with horseradish peroxidase (HRP)-tagged secondary antibodies directed against either mouse or rabbit IgG.

。在4°C下种植过夜后，与对PPAR-γ（稀释度1:1000）或β-肌动蛋白（稀释度1:8000）特异的一抗孵育。用含0.05%吐温20的Tris缓冲盐水冲洗三次，间隔10分钟后，用辣根过氧化物酶（HRP）标记的针对小鼠或兔IgG的二抗处理膜。

For β-actin, an HRP-tagged anti-mouse antibody was applied as a loading control. Protein expression was visualized using an enhanced chemiluminescence substrate and documented with a ChemiDoc XRS imaging system..

对于β-肌动蛋白，将HRP标记的抗小鼠抗体用作上样对照。使用增强的化学发光底物可视化蛋白质表达，并用ChemiDoc XRS成像系统记录。。

RNA isolation and quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Meibomian glands (MGs) were meticulously dissected under a microscope, with the surrounding skin, subcutaneous tissue, muscle, and palpebral conjunctiva being removed. Total RNA was then extracted from these MG samples utilizing the Invitrogen TRIzol reagent (catalog number 15596-018; Thermo Fisher Scientific).

在显微镜下仔细解剖睑板腺（MGs），去除周围的皮肤，皮下组织，肌肉和睑结膜。然后使用Invitrogen TRIzol试剂（目录号15596-018；Thermo Fisher Scientific）从这些MG样品中提取总RNA。

Each experimental group comprised three samples, with each sample being a composite of MG tissues harvested from both eyes of an individual mouse. A consistent quantity of RNA from each sample was converted into complementary DNA (cDNA) through a reverse transcription process, employing a commercially available kit (RR047A; TaKaRa, Shiga, Japan) and adhering to the provider’s guidelines.

每个实验组包括三个样品，每个样品是从单个小鼠的两只眼睛收获的MG组织的复合物。使用市售试剂盒（RR047A；TaKaRa，Shiga，Japan）并遵循供应商的指导原则，通过逆转录过程将每个样品中一致数量的RNA转化为互补DNA（cDNA）。

Subsequently, qRT-PCR was executed with the StepOne Real-Time PCR detection system and a SYBR Premix Ex Taq Kit (catalog number RR420A; TaKaRa), following the manufacturer’s prescribed protocol. The specific primer sequences for the targeted genes are outlined in the accompanying table. The qRT-PCR protocol involved an initial step of denaturation at 95 °C for 1 min, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 30 s.

。目标基因的特定引物序列在附表中概述。qRT-PCR方案涉及在95°C变性1分钟的初始步骤，然后在95°C变性10 s并在60°C退火/延伸30 s的40个循环。

Post-amplification, a melt curve analysis was performed to verify the specificity of the amplification. The relative gene expression levels were determined using the comparative threshold cycle (Ct) method and were normalized against β-actin, which served as an endogenous control gene (Table .

扩增后，进行熔解曲线分析以验证扩增的特异性。使用比较阈值循环（Ct）方法确定相对基因表达水平，并针对作为内源性对照基因的β-肌动蛋白进行标准化（表。

Table 1 Mouse primer sequences used for quantitative RT-PCR.

表1用于定量RT-PCR的小鼠引物序列。

Full size table

全尺寸表

Statistical analysis

统计分析

Data were processed using GraphPad Prism 10.0 software (GraphPad Software Inc, San Diego, CA, USA). Statistical analysis was performed using a Mann-Whitney test with

使用GraphPad Prism 10.0软件（GraphPad software Inc，圣地亚哥，加利福尼亚，美国）处理数据。使用Mann-Whitney检验进行统计分析

< 0.05 considered as statistically significant.

<0.05被认为具有统计学意义。

Data availability

数据可用性

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

本研究中使用和/或分析的数据集可根据合理要求从通讯作者处获得。

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Funding

资金

National Natural Science Foundation of China (Grant No. U20A20363, 81970776, 82301173); The Natural Science Foundation of Heilongjiang Province, China (Grant No. LH2020H039); Heilongjiang Provincial Department of Education Heilongjiang Provincial Higher Education Fundamental Research Project (2021-KYYWF-0226); Heilongjiang Provincial key research and development plan guidance project (GZ20220125); The Science Fund for Excellent Young Scholars of First Affiliated Hospital of Harbin Medical University (Grant No.2024YQ04).

国家自然科学基金（批准号U20A203638197077682301173）；黑龙江省自然科学基金（批准号LH2020H039）；黑龙江省教育厅黑龙江省高等教育基础研究项目（2021-KYWF-0226）；黑龙江省重点研发计划指导项目（GZ20220125）；哈尔滨医科大学第一附属医院优秀青年科学基金（批准号：2024YQ04）。

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作者信息

Authors and Affiliations

作者和隶属关系

Eye Hospital, The First Affiliated Hospital of Harbin Medical University, Harbin, China

Shu Wang, Yulin Li, Jingfan Gao, Jia Lin, Xin Jin & Hong Zhang

王澍、李玉林、高景凡、贾琳、辛进、张虹

Harbin Medical University, Harbin, China

哈尔滨医科大学，哈尔滨

Shu Wang, Yulin Li, Jingfan Gao, Jia Lin, Xin Jin & Hong Zhang

王澍、李玉林、高景凡、贾琳、辛进、张虹

Authors

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Shu Wang

王澍

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Yulin Li

李玉林

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Jingfan Gao

高景凡（音）

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Jia Lin

贾琳

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Xin Jin

辛瑾

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Hong Zhang

张虹（音）

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Contributions

捐款

Author contributionsIn this study, the contributions of the authors were as follows:- Shu Wang, Yulin Lin were involved in the conception and design of the research.- Shu Wang, Yulin Lin, Jingfan Gao and Jia Lin conducted the experiments, data collection, and initial analysis.- Xin Jin were responsible for the statistical analysis and interpretation of the results.- Hong Zhang contributed to the acquisition of funding and supervision of the project.- Shu Wang drafted the initial manuscript and revised it critically for important intellectual content.- All authors read and approved the final manuscript..

作者贡献在这项研究中，作者的贡献如下：-王澍，林玉林参与了这项研究的概念和设计王澍、林玉林、高景凡和贾琳进行了实验、数据收集和初步分析Xin Jin负责对结果进行统计分析和解释张虹为项目的资金获取和监督做出了贡献王澍起草了初稿，并对重要的知识内容进行了批判性修改。。

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Hong Zhang

张虹（音）

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Competing interests

相互竞争的利益

The authors declare no competing interests.

作者声明没有利益冲突。

Ethics approval

道德认可

I can confirm that our study has been conducted in accordance with the ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments).

我可以确认我们的研究是根据ARRIVE指南（动物研究：报告体内实验）进行的。

Additional information

其他信息

Publisher’s note

出版商注释

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。

Electronic supplementary material

电子补充材料

Below is the link to the electronic supplementary material.

以下是电子补充材料的链接。

Supplementary Material 1

补充材料1

Supplementary Material 2

补充材料2

Rights and permissions

权限和权限

Open Access

开放存取

This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material.

本文是根据知识共享署名非商业性NoDerivatives 4.0国际许可证授权的，该许可证允许以任何媒介或格式进行任何非商业性使用，共享，分发和复制，只要您对原始作者和来源给予适当的信任，提供知识共享许可证的链接，并指出您是否修改了许可材料。

You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this licence, visit .

要查看此许可证的副本，请访问。

http://creativecommons.org/licenses/by-nc-nd/4.0/

Reprints and permissions

重印和许可

About this article

关于本文

Cite this article

引用本文

Wang, S., Li, Y., Gao, J.

王，S，李，Y，高，J。

et al.