SCIENTIFIC REPORT

Senescence in cultured trabecular meshwork cells

Yukari Yamazaki¹, Hiroshi Matsunaga¹, Maki Nishikawa¹, Akira Ando¹, Shiho Kaneko¹, Koji Okuda², Mitsumasa Wada¹, Seiji Ito³ and Miyo Matsumura¹

¹ Department of Ophthalmology, Kansai Medical University, Moriguchi, Osaka, Japan
² Department of Paediatrics, Kansai Medical University, Moriguchi, Osaka, Japan
³ Department of Medical Chemistry, Kansai Medical University, Moriguchi, Osaka, Japan

Correspondence to:
Correspondence to:
Dr H Matsunaga
Department of Ophthalmology, Kansai Medical University, 10–15 Fumizono-cho, Moriguchi, Osaka 570-8507, Japan; matsunag{at}takii.kmu.ac.jp

Background: It has been suggested that replicative senescence might be involved in the pathophysiology of age-related diseases.

Aim: To study the process of senescence in trabecular meshwork (TM) cells.

Methods: Porcine TM tissues were obtained and placed in primary cultures with Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium. After 2–3 weeks, migrated and proliferated TM cells were trypsinised and cultured in serial passages, and identified with fluorescein-labelled low-density lipoprotein (DiI-Ac-LDL), a marker of TM cells. Staining for senescence-related ß-galactosidase activity was performed at population doubling level (PDL) 2, 8 and 16 at pH 6. Terminal restriction fragment (TRF) length was examined by Southern blot analysis using a ³²P-labelled telomere-specific sequence (TTAGGG)₃ at each PDL.

Results: DiI-Ac-LDL staining revealed that most (nearly 100%) of the cells in the culture were TM cells, which were flattened in shape and positive for senescence-related ß-galactosidase staining at PDL 16. Reduction of TRF length as a function of population doubling was also shown.

Conclusions: TM cells exhibited characteristics of senescence at PDL 16 in vitro. The results demonstrated that cellular senescence may be related to the pathophysiology of primary open-angle glaucoma.

Abbreviations: PDL, population doubling level; POAG, primary open-angle glaucoma; LDL, low-density lipoprotein; TM, trabecular meshwork; TRF, terminal restriction fragment; RPE cells, retinal pigment epithelial cells

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