Alternation of Lipidomic in the Meibomian Gland After Exposure to Intense Pulsed Light in Patients With Meibomian Gland Dysfunction and Suggestions for Applications of 3PM Medicine


 Importance: Aside from the clinical index, there is no established criterion for assessing the effectiveness of intense pulsed light (IPL) in treating meibomian gland disease.Objective：To determine if there is an association between changes in the meibum lipidomic profiles and alleviation of clinical signs in patients with meibomian gland dysfunction (MGD) who are treated with IPL. To provide predictive, preventive and and personalized medical programs for MGD patients. Design: This is an observational Study. Patients were followed for up for 6 months from January 1, 2019.Setting: This is a single center, human-oriented clinical and basic research study.Participants: Adult patients, who were diagnosed with MGD and had not received any alternative treatments for at least 3 months, were enrolled in the study. Exposures: Each patient received a series of three treatments at 3-week intervals. The meibum was collected before the first treatment (T0) and the third treatment (T2). All enrolled patients completed the whole examination and treatment. The meibum of randomly assigned 26 patients and 10 healthy volunteers was chosen for performing the lipid analysis using LC-MS/MS. Main Measures: The following information from each patient was recorded: tear break-up time (BUT), average tear BUT, tear meniscus height, assessment of the lid margin, bulbar redness, meibomian gland opening position, corneal fluorescein staining, meibomian gland drop, meibomian gland expressibility, and meibum quality.Results: A total of 191 patients’ right eyes were enrolled in the study, including 95 females (49.7%) and 96 males (50.3%), with a median age of 53 years (range: 28–85 years). IPL increased the BUT (P<0.0001, t=7.9, df=380), average tear BUT (P<0.01, t=2.774, df=374.5), and tear meniscus height (P<0.01, t=2.642, df=367.1). At the same time, IPL improved bulbar redness (P<0.0001, t=12.95, df=380) and corneal fluorescein staining (P<0.0001, t=4.147, df=380). Furthermore, following IPL treatment, the meibomian gland expressibility (MGE) and meibum quality were significantly improved (from 1.342±0.05815 to 0.9354±0.03922, P<0.0001, t=5.798, df=380 and from 1.266±0.04969 to 0.8639±0.03318, P<0.0001, t=6.733, df=380, respectively). Lipidomic analysis of the meibum characterized the changes in lipid profiles induced by IPL. Conclusion: IPL treatment offers a novel approach to markedly improve the treatment of patients with MGD due to correction of altered lipid profiles. The changes in lipid expression profiles are potential indexes to evaluate the therapeutic effectiveness of IPL treatment or other treatments on MGD. The lipid changes that are pertinent include: LPC(18:2)+HCOO, OAHFA(18:1/34:1)-H, TG(16:0/18:2/20:5)+H, and etc.. Therefore, accurate evaluation of the changes of lipid expression profile in patients with MGD can be used as a predictive, preventive, and personalized medical method.


Introduction
Dry eye (DE) is becoming the most common ocular surface disease worldwide. As de ned by the International Dry Eye Workshop II, DE is a multifactorial disease of the ocular surface characterized by loss of homeostasis of the tear lm, and accompanied by ocular symptoms. In this condition, tear lm instability and hyperosmolarity, ocular surface in ammation and tissue damage, and neurosensory abnormalities play etiological roles 1  Epidemiological surveys have shown that the prevalence of MGD in Europe has reached 21.9% 4,5 , and that in Asian populations it is as high as 69.3% 6-9 . At present, there is no uniform diagnostic standard for MGD 10 . The criteria for arriving at a diagnosis are not de nitive and limited to determining if: (1) the glandular glands are absent; (2) there are abnormalities between the temporal margin and MGs; and (3) the volume and quality of MG secretions are altered.
Any of the above signs in combination with ocular symptoms are indicative of MGD. Due to the lack of symptomatic speci city assigned to MGD, it is often misdiagnosed and mistreated. Long-term MG lesions can cause in ammatory reactions in the ocular surface, which in turn lead to corresponding changes in the cornea and conjunctiva 11 . In severe cases, visual acuity declines, resulting in poor prognosis. These limitations in arriving at accurate diagnosis is prompting increasing attention by researchers in gaining insight into the pathophysiology underlying MGD.
Intense pulsed light (IPL) is a noncoherent polychromatic light source with a broad wavelength spectrum of 500-1,200 nm 12,13 . As an established commercial technology, IPL treatment is broadly used in diseases involving facial rosacea. It has been shown that IPL is effective for the treatment of the eyelid sebaceous gland, also termed MG [14][15][16] . Thus, IPL is a promising new therapy for MGD. Prior to the development of IPL, the treatment against MGD or DEs involved warming and massage 17 . Currently, IPL treatment is regarded as a more time-e cient and effective method to ameliorate the symptoms of DE 18 . Although the e cacy and safety of IPL had been con rmed 19 , the most signi cant changes in the rouge composition before and after exposure to IPL remain unclear.
The palpebral lipids are mainly composed of wax lipids, cholesterol lipids, free fatty acids, phospholipids, and a small amount of protein. More than 100 components of palpebral lipids have been found, and hundreds of subtypes have not been identi ed thus far [20][21][22] . In this study, a comprehensive evaluation of clinical signs and changes in the lipid composition of meibum was conducted in patients with MGD after at least three exposures to IPL. A comparison of clinical assessment and alteration of the meibum was also conducted in patients treated with IPL and those without MGD. The results of this study will be helpful in understanding the mechanisms through which IPL can effectively treat MGD, besides providing insightful data for future studies. Through our research, we can provide predictive, preventive and and personalized medical services for MGD patients.

Patients
Adult patients, who were diagnosed with MGD and had not received any alternative treatments for at least 3 months, were enrolled in the study. The inclusion criteria were: diagnosis of MGD according to the Japanese MGD diagnostic criteria (Bron and Tiffany 2004), including ocular symptoms, plugged gland ori ces, vascularity of lid margins, irregularity of lid margins, and decreased meibum quality and quantity. The exclusion criteria were: previous ocular surgery or trauma; blepharal dysraphism; history of blepharal and periorbital skin disease within 1 month prior to enrollment; acute in ammation; rheumatic immune systemic diseases; excessive sun exposure within 1 month prior to enrollment; history of herpes zoster infection; pregnancy; and use of photosensitive drugs/foods. Informed consent was provided by all patients following explanation of the nature and possible consequences of the study. This study was approved by the Institutional Review Board of the Xin Hua Hospital of Shanghai Jiao Tong University School of Medicine (Shanghai, China) and was registered with the Chinese Clinical Trial Registry prior to enrollment of the rst patient. This study adhered to the tenets of the Declaration of Helsinki. All examiners were blinded to the treatment group. The study was enlisted in the clinical trial registry (trial registration no.: ChiCTR2000033454).

IPL treatment
Each patient received a series of three treatments at 3-week intervals. The patients had to use Proparacaine Hydrochloride Eye Drops at least twice prior to each treatment. A modular laser multi-application platform (Quantum™, Lumenis, USA) was used to administer treatment to the periorbital area. During the IPL procedure, patients were required to wear opaque goggles, and the coolant was applied around the eyelids under both eyes. Makeup and contact lenses were removed before treatment. Depending on patient tolerance, each subject in the patient group received 2-3 light pulses. The intensity of IPL treatment (12~15 J/cm 2 ) depending on the Fitzpatrick skin with a 590-nm lter.

Clinical assessment
Visual acuity and intraocular pressure were recorded in the case-report form. The Standard Patient Evaluation of Eye Dryness questionnaire was completed by each patient. Schirmer 1 test, corneal staining, meibum quality and meibomian gland expressibility (MGE), lid margin abnormality, bulbar redness, MG opening position, and corneal uorescein staining were measured through slit lamp microscopy. A noncontact infrared meibography system was used to assess tear BUT, MG dropout, and tear meniscus height.

Meibum acquisition
The use of makeup, arti cial tears, and cleaning the rim more than 3 hour prior to the collection of the material was prohibited. After exposure to IPL, proparacaine hydrochloride eye drops were used again and the material was collected. The use of plastic products during sample preparation and experimentation was avoided to prevent contamination of the rouge. The steps performed for the collection of the materials were as follows: 1) the MG pressing method was used to squeeze out the secretions of the MGs with a MG expressor forceps -this is achieved by placing the thumb of one hand under the lower eyelid, squeezing the rim and rapidly pulling down, and squeezing out the secretion of the MG (the same method was used to remove the rouge from the upper eyelid); 2) a medical cotton swab was used to scrape the MG secretions, the cotton swab was placed it in a cryotube, and stored at −80°C until further analysis. The amount of rouge collected in this study was 1.77±0.29 mg.
Ultrahigh pressure liquid chromatography liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) LC separation using CSH C18 column (1.7 µm, 2.1 mm × 100 mm, waters) was selected to perform reverse phase chromatography. The lipid of meibum was re-dissolved in 200 µL 90% isopropanol/ acetonitrile, than centrifuged at 14,000 xg for 15 min, nally 3 µL of the sample was selected for injection. Solvent A: acetonitrile-water (6:4, v/v) with 0.1% formic acid and 0.1Mm ammonium formate. Solvent B: acetonitrile-isopropanol (1:9, v/v) with 0.1% formic acid and 0.1mm ammonium formate. The initial mobile phase was 30% solvent B at a ow rate of 300 μL/min. It was held for 2 min, and then linearly increased up to 100% solvent B in 23 min, followed by equilibrating at 5% solvent B for 10 min. Mass spectra was acquired by Q-Exactive Plus in positive and negative mode, respectively. ESI parameters were optimized and preset for all measurements as follows: Source temperature, 300 °C; Capillary Temp, 350 °C, the ion spray voltage was set at 3,000V, S-Lens RF Level was set at 50% and the scan range of the instruments was set at m/z 200-1800. "Lipid Search" is a search engine for the identi cation of lipid species based on MS/MS math. LipidSearch contains more than 30 lipid classes and more than 1,500,000 fragment ions in the database. Both mass tolerance for precursor and fragment were set to 5 ppm.

Statistical analysis
Data were analyzed using the SPSS version 22.0 (IBM Corp., Armonk, NY, USA) software. Continuous intergroup variables were analyzed using an independent t-test, and pretreatment and continuous intragroup variables were analyzed with a paired t-test. Categorical intergroup variables were analyzed with the nonparametric Kruskal-Wallis test, and intragroup analysis of categorical variables was performed using the nonparametric Wilcoxon signed-rank test. Correlations between normally distributed values and non-normally distributed values were analyzed with the linear Pearson correlation coe cient and the Spearman correlation coe cient respectively. P<0.05 denoted statistical signi cance.

Patient demographics
A total of 191 patients' right eyes were enrolled in the study, including 95 females (49.7%) and 96 males (50.3%), with a median age of 53 years (range: 28-85 years). We collected the meibum before the rst treatment (T0) and the third treatment (T2) All enrolled patients completed the whole examination and treatment; there was no pain or any discomfort reported during the tests. Among the patients, we chose 26 patients randomly to do the lipid analysis using LC-MS/MS. And we also recruited 10 volunteers who had no ocular surface disease and other systemic disease (The work plan is illustrated as follows) ( Table 1).

MGE and meibum quality
The scores of MGE and meibum quality were used to evaluate the quality and expressibility of the meibum. Following IPL treatment, the meibum changes from solid state to liquid state and becomes clear and easy to eliminate, the MGE score and meibum quality score were signi cantly decreased (from 1.34±0.06 to 0.94±0.04, P<0.0001, t=5.798, df=380 and from 1.27±0.05 to 0.86±0.03, P<0.0001, t=6.733, df=380, respectively) ( Table 2).

Lipid metabolism of MG
As previously reported, IPL shows great therapeutic potential for patients with MGD and ocular surface diseases. It is well established that MGs are rich in lipids. The meibum and lipids of the MG were collected to perform a lipid analysis using LC-MS/MS. A total of 323 lipid species were identi ed and most of them were rich in triglyceride (TG, n=127), ceramide (Cer, n=86), phosphatidylcholine (PC, n=29), sphingomyelin (SM, n=28), simple Glc series G1 (CerG1,  (Fig. 1A). When we compared the T0 to control and T2 to T0, there were signi cant differences observed between the groups in lipid quality, as shown by the volcano plot (Figs. 1B and C). Moreover, when we compared the T2 to T0, most of the different lipids were decreased in the former; when we compared the T0 to control group, most of the different lipids were increased in T0. At the individual lipid compound level, the increased lipids in T0 (i.e., CerG1, CerG2, Co, DG, LPC, LPE, LPG, LPI, LPS, OAHFA, StE, and TG) were decreased following IPL treatment (T2) (Figs. 2A1-A12). There were no signi cant differences in Cer, DGDG, LSM, MGDG, PG, PI, PS, SM, SO, and WE that increased in T0 and T2 (Figs. 2B1-B10). The decrease in DGMG and MGMG in T0 could be reversed by IPL treatment (T2) (Figs. 2C1 and C2). Furthermore, the levels of PC and PE in T2 were higher than those measured in T0 (Figs. 2D1 and D2).  Table 4). The change of the lipid metabolic pattern was highly related to the improvement of the clinical index (Fig. 3). The  (Table 5). We found that Cer(d18:1/18:0)+HCOO (C 37 H 72 O 5 N 1 ) was decreased in T0, whereas it was increased in T2 (Table 5) (Fig. 4).

Discussion
This is the rst study describing the effects of IPL treatment on the lipidomics analysis of the meibum after treatment with IPL in MGD. Based on the changes induced by IPL treatment, such changes are associated with reversal of different symptoms characteristic of meibomian gland disease. This treatment led to improvement of the ocular surface health resulting from increasing tear production, stabilizing the tear lm, improving corneal epithelial cell contacts, and decreasing the conjunctival in ammation. Interestingly, IPL treatment also improved the quality and expressibility of the meibum from the meibomian glands. Therefore, IPL treatment provides a novel approach that may improve management of meibomian gland diseases in a clinical setting. IPL can be used as a predictive, preventive and presonalised medical scheme for the treatment of MGD patients.
IPL is a popular procedure for treating skin and ocular diseases. The IPL devices employ xenon gas-lled ash lamps to produce non-laser high-intensity pulses of polychromatic non-coherent lights in a broad-wavelength spectrum, from visible (515 nm) to infrared (1,200 nm). The mechanism of action of IPL systems is based on the principle of selective thermolysis, according to which certain targets, termed chromophores, are capable of absorbing and subsequently converting through non radiative transfer the impinging light into heat energy. IPL treatment has been utilized for years in dermatological clinical practice 23,24 . Recently, this procedure was also applied in ophthalmology to treat MGD 25,26,27 . Its use for this purpose stemmed from declines in DE symptoms noted in patients who underwent IPL treatment for facial rosacea. Previous research studies con rmed the e cacy of IPL treatment in relieving the symptoms and signs of MGD 26,27 .
In our study, the patient IPL treatment resulted in marked increases in both the BUT and aBUT values. As previously reported, these rises occurred as a result of improved tear lm stability and a reduction in the tear evaporation rate. These effects are in accord with our measurement of a signi cant increase in tear production. Furthermore, we found that the ocular surface health improved based on declines in bulbar redness and corneal uorescein staining.
IPL treatment also reduced capillary formation on the ocular surface which is advantageous to the restoration of corneal surface health. In addition, it is known to inhibit the formation of brown spots, and sun damage (excluding skin cancer) on the face, neck, chest, arms, and hands 28 . Another previous study showed that IPL improved the ori ce abnormality in MG 18 . In our study, although the IPL improved the quality and expressibility of the meibum, this procedure did not improve the ori ce abnormality in MG.
The meibum is rich in lipids 21 . We hypothesized that the recorded improvement in the quality and expressibility of the meibum was due to an alternation of the meibum lipidomic pro le. Based on the effects of IPL treatment on the principal component analysis of MGD patients, this procedure reversed a number of changes in the lipidomic pro le towards patterns characteristic of those seen in healthy subjects. The moderate declines induced by IPL treatment in the MGD symptomology were associated with reversal of the hyperlipidemia in 12 of the 27 classes of lipids. It is possible that the restoration of the normal healthy pro les in this group of lipids contributed to suppression of MGD symptomology 18 . Of note, IPL treatment did not alter any of the other 15 classes of lipids changed by MGD. Furthermore, two sub classes (PC and PE) were higher than those in the untreated MGD group. Collectively, these results indicate that IPL treatment cannot completely reverse MGD symptomology, which is in accord with the failure of this procedure to fully reverse all of the changes in the lipidomic pro le induced by MGD.
Despite the reversal of the changes in an appreciable number of lipid classes by IPL treatment, there were an appreciable number of insensitive lipid classes that could underlie failure of the treatment to fully reverse all of the MGD symptomology. Nevertheless, we found that the increases in 13 subclasses of lipids and decrease in one subclass of lipids in MGD could be reversed by IPL. They were rich in OAHFA, LPC, TG, and Cer. These lipids may be suitable markers for monitoring responses to IPL treatment. BUT, CR and meibum quality are three clinical indices that were vindicated as being relevant markers to assess IPL treatment e cacy.
Lastly, we found that IPL treatment could reverse some of the MGD-induced hyperlipidemia. These changes alleviated some of the MGD symptomology through stabilizing the tear lm and improving the ocular surface health. Therefore, IPL can be used as an effective regimen for predictive, preventive and personalised in the treatment of MGD.

Declarations
Consent to participate: Informed consent was provided by all patients following explanation of the nature and possible consequences of the study. Tables   Table 1. A working diagram of data processing Note: BUT, break-up time; aBUT, average break-up time; CR, redness of conjunctival (CR) in ammation; FL, corneal uorescein staining; MGE, meibomian gland expressibility. ** P<0.01 ****P<0.0001.     The correlation of signi cant subclasses of lipids with clinical syndrome. The change of the lipid metabolic was high related the improvement the clinical index. The correlation was rich in BUT1, CR and meibum quality and some lipids were cross relation with them. The green in the middle represents the clinical index data, and the orange in the outer circle is the differential metabolic lipid. The redder the connection between them, the closer