建立以PM 2.5誘導的急性過敏性結膜炎小鼠動物模型

一些流行病學證據顯示出直徑小於或等於2.5 um的細懸浮微粒(PM 2.5)可能是過敏性結膜炎的誘發原因之一，但目前尚未有實驗數據可以說明其機制。為闡明其誘發機制，本研究以小鼠作為研究模型，將實驗分為兩階段。第一階段先建立適用於小鼠的淚液結晶測試方法，加強對淚液品質的分析。從24隻ICR小鼠收集淚液檢體，在眼睛表面分別用2、5、10或20 ul沖洗液(0.9％ NaCl或無菌水)反覆沖洗後，收集淚液檢體，在24±2℃的溫度條件下測試相對濕度分別為46±2％或53±2％時對淚液結晶的影響，此外還測試了減少雜質 (4000 rpm離心10分鐘)、不同滲透壓(280-360 mOsm/L)和pH值(6.5-8.0)對結晶形成的影響，並評估重複性和乾燥淚液的儲存穩定度。第二階段將溶於0.9％ NaCl 的PM 2.5直接滴在小鼠眼睛表面，以淚液品質及組織病理分析直接接觸PM 2.5是否可引起過敏性結膜炎。將50隻ICR小鼠隨機分組，分別將濃度3.2、6.4和12.8 mg/ml的PM 2.5溶液滴在各組小鼠眼表，一天三次，連續18天，並以0.9％ NaCl溶液同樣滴於小鼠眼表作為對照組，另外，在高劑量組(12.8 mg/ml)額外使用人工淚液作為緩解組。實驗前後分別進行過敏性結膜炎臨床指標評估、淚液量、淚膜破裂時間、抓癢行為及淚液結晶測驗，並在實驗結束時分析角膜健康狀況，並收集整個眼睛和瞼板腺組織，以HE染色、PAS染色及Giemsa染色後，進行組織型態、結膜杯狀細胞以及嗜酸性細胞浸潤程度分析。第一階段實驗結果確定適合淚液結晶條件為以2 ul 0.9％ NaCl作為沖洗溶液，在相對濕度為46±2％下時可得到較典型的淚液結晶結果。除此之外，離心檢體並沒有得到較好的結晶結果，且乾燥結晶的數據紀錄最好在十天內完成。較高滲透壓的溶液結晶狀況變差，而pH值在6.5-8.0之間結晶的型態沒有明顯變化。在第二階段結果中，臨床指表評分顯示在第18天時，眼瞼浮腫、泛淚和抓癢行為會隨著PM 2.5濃度增加而呈現正相關性，淚液量增加且淚液破裂時間更長。淚液結晶試驗顯示，暴露於PM 2.5後，晶體的結構愈來愈差，甚至無法形成結晶。PM 2.5處理後上結膜杯狀細胞數量上升，並且結膜和瞼板腺嗜酸性細胞浸潤數量增加。本研究結果除建立小鼠淚液結晶測試方法外，並確定小鼠眼睛表面接觸PM 2.5後會誘發類似於臨床上過敏性結膜炎的症狀。

Several epidemiologic evidences have indicated that PM 2.5 (particulate matter with a diameter of 2.5 lm or less) as a causing factor of allergic conjunctivitis. However, no experimental data is currently available to elucidate the mechanism induced by PM 2.5 in mice. In this study, the first stage aimed to establish a mouse tear ferning test protocol for tear quality analysis. Tear samples were collected from 24 healthy mice after repeated flushes the ocular surface with 2, 5, 10, or 20 μl wash solutions, either using 0.9% NaCl saline or sterile water. After sample collection, tear ferning tests were performed at a relative humidity of either 46± 2% or 53±2%, and with temperature fixed at 24±2℃ for comparison. Moreover, the influence of osmolarity (280-360 mOsm/L) and pH values (6.5-8.0) and the effect of centrifugation (4000 rpm, 10 minutes) on ferning formation were examined. Reproducibility and ferning storage stability were also determined. The second stage investigated whether PM 2.5 solution directly applied to the mouse ocular surface might induce allergic conjunctivitis, and analyzed its underlying mechanisms. 50 ICR mice were subject to eye drops containing PM 2.5 at 3.2, 6.4, and 12.8 mg/ml in 0.9% NaCl saline for 18 consecutive days, along with the controls prepared in parallel without PM 2.5 and another control group treated with both PM 2.5 at 12.8 mg/ml and artificial tears. The whole eyes and Meibomian glands were harvested for histopathological analyses and assessment of clinical scoring, tear volume, tear breakup time, and tear ferning. Furthermore, goblet cells by periodic acid Schiff stain and infiltrated eosinophil by Giemsa stain were quantified and compared among study groups. An optimized protocol was established at the first stage with relative humidity at 46±2% and drop aliquot at 2 μl 0.9% NaCl saline. Centrifugation did not improve ferning formation, and the tear ferning, once formed, was stable only for ten days under room temperature and environment. Higher osmolarity increased tear ferning grading scores, but pH values did not affect ferning formation. At the second stage, clinical scoring showed more eyelid edema, tearing, and scratching behavior, with longer tear breakup time under the influence of increased PM 2.5 concentrations. Tear ferning test showed worse ferning formation after exposure to PM 2.5. In addition, more goblet cells and eosinophils accumulated in the upper palpebral conjunctiva and in the Meibomian glands. In conclusion, this study established the first mouse tear ferning protocol and demonstrated that, when the ocular surface was directly exposed, PM 2.5 can induce symptoms similar to those found in clinical allergic conjunctivitis.