Pisella Pierre-Jean, Debbasch Caroline, Hamard Pascale, Creuzot-Garcher Catherine, Rat Patrice, Brignole Françoise, Baudouin Christophe
Department of Ophthalmology, University Hospital of Tours, Tours, France.
Invest Ophthalmol Vis Sci. 2004 May;45(5):1360-8. doi: 10.1167/iovs.03-1067.
To compare the toxicity of latanoprost and preserved and unpreserved timolol on conjunctival cells. Expression of inflammatory markers and MUC5AC-related mucin production were evaluated by impression cytology in a case-control ex vivo study. The proapoptotic effect of the same drugs was also evaluated in vitro in a conjunctival cell line and compared with that of benzalkonium chloride (BAC).
Impression cytology (IC) specimens were obtained from a series of normal subjects and from patients with glaucoma treated for at least 1 year with latanoprost eye drops or preserved or unpreserved timolol. All groups were comparable in age and duration of treatment. Expression of HLA-DR, intercellular adhesion molecule (ICAM)-1, and mucin was evaluated in a masked manner by flow cytometry. For the in vitro study, a human conjunctiva-derived cell line was treated with 0.02% BAC-containing latanoprost or timolol, unpreserved timolol, or 0.02% BAC alone for 15 minutes, followed or not by 4 or 24 hours of cell recovery in normal medium. Cell viability and chromatin condensation were evaluated using microplate cold light cytofluorometry with the neutral red and the Hoechst 33342 tests, respectively. The Hoechst-neutral red ratio was defined for the apoptosis assay, and cytoskeleton changes were assessed by confocal microscopy.
No difference was found between normal eyes and those receiving unpreserved timolol. Preserved latanoprost and timolol significantly increased the inflammatory marker expression and decreased MUC5AC expression, but to a significantly higher extent in the preserved timolol group compared with latanoprost. In vitro, 0.02% BAC-containing timolol and latanoprost triggered conjunctival cell apoptosis-however, to a significantly lesser extent than did 0.02% BAC alone. Unpreserved timolol did not cause any cell toxicity.
These ex vivo and in vitro studies demonstrate that BAC-containing latanoprost and timolol exhibit higher proinflammatory and proapoptotic effects on conjunctival cells than does unpreserved timolol. Latanoprost caused less toxicity, however, than preserved timolol, and both drugs were less toxic than BAC alone. These results suggest a potential protective effect of the prostaglandin analogue and to a lesser extent of timolol against the toxicity of BAC in conjunctival cells.
比较拉坦前列素以及含有防腐剂和不含防腐剂的噻吗洛尔对结膜细胞的毒性。在一项病例对照离体研究中,通过印迹细胞学评估炎症标志物的表达以及与MUC5AC相关的黏蛋白生成。还在体外对结膜细胞系评估了相同药物的促凋亡作用,并与苯扎氯铵(BAC)进行比较。
从一系列正常受试者以及使用拉坦前列素滴眼液或含有防腐剂或不含防腐剂的噻吗洛尔治疗至少1年的青光眼患者中获取印迹细胞学(IC)标本。所有组在年龄和治疗时长方面具有可比性。通过流式细胞术以盲法评估HLA-DR、细胞间黏附分子(ICAM)-1和黏蛋白的表达。对于体外研究,将人结膜来源的细胞系用含0.02%BAC的拉坦前列素或噻吗洛尔、不含防腐剂的噻吗洛尔或仅用0.02%BAC处理15分钟,随后在正常培养基中恢复4或24小时(恢复与否)。分别使用中性红和Hoechst 33342试验通过微孔板冷光细胞荧光测定法评估细胞活力和染色质凝聚。定义Hoechst-中性红比率用于凋亡测定,并通过共聚焦显微镜评估细胞骨架变化。
正常眼睛与接受不含防腐剂噻吗洛尔的眼睛之间未发现差异。含有防腐剂的拉坦前列素和噻吗洛尔显著增加炎症标志物表达并降低MUC5AC表达,但与拉坦前列素相比,含有防腐剂的噻吗洛尔组程度显著更高。在体外,含0.02%BAC的噻吗洛尔和拉坦前列素引发结膜细胞凋亡——然而,程度明显低于单独使用0.02%BAC。不含防腐剂的噻吗洛尔未引起任何细胞毒性。
这些离体和体外研究表明,与不含防腐剂的噻吗洛尔相比,含BAC的拉坦前列素和噻吗洛尔对结膜细胞表现出更高的促炎和促凋亡作用。然而,拉坦前列素的毒性低于含有防腐剂的噻吗洛尔,并且两种药物的毒性均低于单独使用的BAC。这些结果表明前列腺素类似物以及在较小程度上噻吗洛尔对结膜细胞中BAC的毒性具有潜在保护作用。