Effect of light-emitting diodes with different color rendering indexes on the ocular tissues of rat

INTRODUCTION

In 2015, the International Dry Eye Working Group recognized that dry eye disease (DED) is a multifactorial ocular surface disease

. The loss of tear film homeostasis and ocular surface symptoms are its main characteristics,insufficient water type is one of the common subtypes of dry eye

. Studies have shown that inflammation is one of the key factors in the pathogenesis of dry eye

. Lacrimal gland is the main place for secretion of tear fluid. Inflammation of the lacrimal gland will seriously affect the composition of the tear film and produce DED. A variety of inflammatory factors have been confirmed to be involved in the pathology of dry eye, among which tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) play an important role

. They are both core members of the cytokine network, IL-6 is a cytokine that regulates inflammation and an important regulator of B cell and T cell functions. In patients with dry eye, multiple inflammatory factors increase in the conjunctival epithelium,and IL-6 is the most valuable

.

The LED-L group had the thinnest thickness of about 116.2±11.72 μm, which had a statistical difference compared with the control group (

＜0.05). The NFL has sparse and disordered arrangement of nerve fibers; the number of ganglion cells were decreased, and the nuclei were small and round;the number of cell-layers of INL was significantly decreased and the distribution of cells were sparse; the OPL was thin and partially disappeared; the number of ONL cells was only 1-2,and the cells were scattered; the photoreceptor layer of rod and cone was seriously thinned, and the thickness was consistent with RPE’s; the choroidal blood vessel density was increased.In the LED-M group, the thickness of the retina was about 128.8±7.65 μm which was significantly lower than the control group (

＜0.05) but higher than the LED-L group (

＜0.05).Some of the nerve fibers in the NFL had disappeared; some cells in the GCL became uneven staining; the INL, which thickness was increased, had abnormal cells protruding into the IPL, uneven nucleoplasm staining, chromatin edge clustering,and sparse distribution of cells; the OPL had a reduced thickness and was discontinuous; the ONL had about 3-4 layers of cells with loose distribution; the ELM was uneven;the cells of photoreceptor layer of rod and cone were sparsely distributed, the thickness of this layer increased slightly and obvious with the sparse arrangement; the choroid was obviously thickened with dense blood vessels.

The emergence of light-emitting diodes (LEDs) has a revolutionary impact on the development of artificial light sources. Living for a long time in the LEDs exposure environment will cause irreversible phototoxicity to eye

,such as damaging corneal epithelial microvilli and then destroying tear film stability, inflammation and degeneration of the retina,

. The color rendering index (CRI) is an index comprised between 0 and 100, defining the ability of a light source to reproduce the various colors of objects illuminated by it when compared to a reference light source

. By definition, daylight has a CRI of 100. In the lighting industry,Ra is used to quantitatively evaluate the CRI of the light

.It’s reported that a light source with a larger CRI is closer to natural light and has better visual comfort

. So, since the CRI is related to visual comfort, will it affect the occurrence of dry eye and the morphological changes of eye? In this study, rats were exposed to three LEDs with different CRIs to observe dry eye indicators, lacrimal gland and retinal pathological changes. The protein expression levels of TNF-α and IL-6 in the lacrimal gland were assessed

immunofluorescence. We aim to support the practicality and feasibility of this model,and to provide a certain degree of experimental basis for the optimization of the new LED from the animal experiment level.

MATERIALS AND METHODS

最令人擔心的是，根據愛因斯坦的理論，這顆以每秒5000英里的速度沿著雞蛋形軌道疾馳的恒星，應該經歷了宇宙中的所有奇異之處。這顆恒星表面遭受的強烈引力會減緩光波的振動，將其拉長。于是，從地球上看來，它會變得比正常狀態更紅一些。

因此，在臨床膿毒癥救治過程中，需要高度重視患者血小板的功能變化，積極采取有效的干預措施，從而提升膿毒癥患者的治愈率。2012版嚴重膿毒癥/膿毒性休克治療指南指出，輸注血小板的指導原則來源于專家共識意見和化療引起的血小板減少癥的經驗[3]。陳樸等[15]的研究結果顯示重組人血小板生成素治療能夠有效促進膿毒癥患者血小板計數恢復，減少患者輸注濃縮血小板等血制品的數量，降低患者的病死率。

Schirmer I Test Schirmer I test (SIt) was performed at the beginning of the experiment, 2 and 4wk after the experiment.According to the standard of Fujihara

, a 1×17-mm

size filter paper strip (Tianjin Jingming New Technology Development Co., Ltd., China) was used to measure the amount of tears produced over 2min. The strip was placed in the lateral canthus of the eye. The rats were operated to keep their eyes closed during the course of the test. After removal,the lengths of color change on the trips were measured under a microscope and recorded in millimeters. Repeat 3 times for each eye and take the average.

Statistical Analysis Summary data conforming to the normal distribution were report as means±standard error of mean(SEM) and

value ＜0.05 was regarded as the standard for statistical significance. Multivariate repeated measurement analysis of variance was applied to compare the SIt, BUT,and CFS scores among different groups, and then further comparison was applied by the Student’s

-test. A one-way analysis of variance was used to compare the TNF-α and IL-6 expression among different groups. All parametric statistical analyses were performed on SPSS Statistics 23 (SPSS, Inc.,An IBM Company, based in Chicago, IL, USA) and GraphPad Prism 8.0 (GRAPHPAD Software, Inc., San Diego, CA, USA).

Corneal Fluorescein Sodium Staining The staining of the cornea was performed to assess the degree of corneal damage observed under the cobalt blue light of the slit-lamp microscope. Dip the fluorescein sodium test paper with 1 drop of normal saline, lightly touch the inner side of the lower eyelid of the rat and wait for 3min to make the fluorescein sodium evenly distributed. With reference to the method of Koh

, the scoring standards are as follows: the cornea is divided into four quadrants and scored separately, and the scores are added to form the final score. A score of 0 suggested an absence of fluorescein staining, a score of 1 suggested the slight punctate staining was less 30, a score of 2 suggested the punctate staining was exceeded 30 but there were no flakes, a score of 3 suggested there had severe diffuse staining but no plaque, and a score of 4 was given when plaques of fluorescein was appeared. Corneas were examined every two weeks beginning the first day.

Hematoxylin-Eosin Staining After the experiment, the rats were killed by intraperitoneal injection of 10% chloral hydrate.The lacrimal gland and retina were taken out and fixed in 4%paraformaldehyde and FAS eyeball fixation solution (G1109-50ML, Wuhan Servicebio Technology Co., Ltd., Wuhan,China) for 72h. The tissues were dehydrated with gradient alcohol, transparent xylene, and paraffin embedding. Finally, a 4-micron thick tissue specimen was obtained using a paraffin slicer for hematoxylin-eosin (HE) staining. Observation and collection of images was under a 400× optical microscope.

Immunofluorescence Staining After light exposure, the expression of TNF-α and IL-6 in the lacrimal gland were assessed by immunofluorescence staining. Deparaffinize and rehydrate the slices, antigen retrieval, goat serum blocked, anti-TNF-α antibody and anti-IL-6 antibody (GB11188, GB11117,Servicebio) were added dropwise. All sections were incubated overnight in a humid box at 4°C, washed with phosphate buffer saline (PBS) solution three times the next day and then incubated with fluorescent secondary antibody (GB23102,Servicebio) in the dark. The 4’,6-diamidino-2-phenylindole(DAPI; G1012, Servicebio) counterstained the cell nucleus,added autofluorescence quencher after avoiding light at room temperature. Representative images were viewed and captured using an Ortho-Fluorescent Microscopy (Nikon Eclipse C1;Nikon, Japan), and Image J was used to calculate the average fluorescence intensity.

Break-up Time The tear film break-up time (BUT) was measured at the beginning of the experiment, 2 and 4wk after the experiment.Dip the fluorescein sodium test paper (Tianjin Jingming New Technology Development Co., Ltd., China) with 1 drop of normal saline, lightly touch the inner side of the lower eyelid of the rat and wait for 3min to make the fluorescein sodium evenly distributed. BUT was recorded (in seconds) when the first black dry spot appears on the cornea under the cobalt blue light of a slit-lamp microscope (SL-7, Sun Kingdom, Chongqing,China). Repeat 3 times for each eye and take the average.

RESULTS

Histopathology of Lacrimal Gland The normal lacrimal glands in the control group showed round, oval or irregular acinus with complete structure and uniform cytoplasm,which were tightly arranged. The cytoplasm of acinar cells were basophilic, the nucleus were round and varying in size.The cells were stained deeply which could visibly see the chromatin aggregation in the nucleolus. The lobules of the lacrimal gland in the LED-L group shrank and merged withthe loose arrangement; the gland cavities were expanded and had vacuoles; the intracellular eosinophil granules increased significantly; the acinar cells had sparse distribution and different morphology. In the LED-M group, the lobules of the lacrimal gland were atrophied, the gland cavity were expanded and there were a large number of vacuoles, the intracellular eosinophilic granules increased, the irregular nucleoli were scattered meanwhile. Compared with other groups, the lobules of the lacrimal gland in the LED-H group were neatly structured, tightly arranged, and the structure was complete;the cytoplasm were uniform, the cytoplasmic eosinophilia was slightly increased; the nuclei of acinar cells were in different sizes, and the chromatins within the nucleolus were clearly aggregated (Figure 3).

Tear Break-up Time After Light Exposure for 2 and 4wk The BUT of the LED-L group (11.60±0.46s) and the LED-M group (12.88±0.31s) were shorter than that of the control group(14.08±0.28s) after 2wk (

＜0.05), and the difference between these two groups were statistically significant (

＜0.05); But there was no statistically significant difference between LED-H group and the control group. After 4wk, the BUT of the all light exposed groups were shortened again, the BUT of the LED-L, LED-M, and LED-H groups decreased to 6.82±0.34s,9.60±0.77s and 12.74±1.07s, which showed the statistical significance of the differences compared with the control group (14.44±0.73s,

＜0.05). At the same time, there were statistically significant differences between LED-M, LED-H,and the LED-L groups (Table 2, Figure 1B).

The LED-H group, with a thickness of about 131.0±4.758 μm,was similar to that of the LED-M group and was significantly lower than the control group (

＜0.05) but higher than the LED-L group (

＜0.05). While it was more regular and more flat when compared to the LED-M group; the ONL had about 3-4 layers of cells, and the cells were polygonal, sparsely distributed; the photoreceptor layer of rod and cone was tightly arranged; the choroid thickness was close to that of the LED-M group, which had abundant blood vessels. The comparison of the retinal thickness is showed in Figure 5.

Schirmer I Test Scores After Light Exposure for 2 and 4wk In the Schirmer’s test, no significant difference among the four groups was observed before the light exposure. As time increases, the Schirmer’s test scores of the LED-L, LED-M,and LED-H groups decreased to 6.86±0.48, 8.12±0.48, and 8.84±0.77 mm respectively after 2wk, while the control group was 10.42±0.69 mm, and the differences between the exposed group and the control group were statistically significant(

＜0.05). Compared with the LED-L group, the LED-M group and the LED-H group had more tear secretion which made the differences statistically significant (

＜0.05). After 4wk, the SItscores of the LED-L group (4.34±0.82 mm) were statistically lower than that of the LED-H group (8.92±0.56 mm)and LED-M group (7.20±0.78 mm,

＜0.05). Meanwhile, the difference between the LED-M group and the LED-H group was statistically significant (

＜0.05). With the increase of LEDs exposed time, the tear secretion of the LED-L group and the LED-M group decreased markedly (Table 1, Figure 1A).

使用水力清淤混合器可以清除河道淤泥，效果比較好。要確定清淤器的水噴嘴和喉管的比例，噴嘴和吸泥管的比例范圍，需在一定的標準范圍內才行。像喉管橫截面的長度和直徑要相同，才能保證混合泥漿處于穩的狀態；擴散管需做成錐形，減少能量轉化時的能量損失，提高泥漿的位能；泥漿吸入時需要于泥漿相等量的高壓水，保證充分的混合稀釋，保證泥漿的流速；保證噴嘴處的高壓水流速適宜，水壓比例在噴嘴水壓的有效值內。一般來說，清淤機的工作效率并不高，不到水泵效率的的一半。10%-40%。

Histopathology of Retina The morphology of the retina of the rats was showed in Figure 4. There were complete overall structure in all groups, but each characteristics were explained below. In the control group, the retina layers were distinct and tight, and the thickness of the retina was about 174.0±7.601 μm. A clear inner limiting membrane (ILM) can be seen; the nerve fibers in the nerve fiber layer (NFL) were arranged well; the nuclei in the ganglion cell layer (GCL) were mostly oval and dense; the inner plexiform layer (IPL) had intact structure; the inner nuclear layer (INL) nuclei were arranged neatly , mostly round and evenly stained; the outer plexiform layer (OPL) had a clear and complete structure; the outer nuclear layer (ONL)nuclei were tightly arranged, oval, darkly stained and about 9-10 layers; the external limiting membrane (ELM) was clear and complete; the cells of the photoreceptor layer of rod and cone were tightly arranged; the retinal pigment epithelium(RPE) was normal in shape, arranged in a short cubic monolayer, and the cytoplasm contained pigment; the blood vessels in the choroid which contained pigment cells were clearly visible, and the boundaries of them could be seen obviously.

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