Necrosis Is the Primary Mode of Stromal Cell Death Induced by the Femtosecond Laser: Don't Apply the Tunel Assay in Isolation

Abstract

Purpose:: To examine the mode of stromal cell death associated with the femtosecond laser (FS) compared with the microkeratome in LASIK flap formation.

Methods:: Thirty-nine New Zealand White rabbits were divided into groups that had corneal flaps formed with a 15 KHz FS laser (Intralase, Irvine, CA), a 30 KHz FS laser, a 60 KHz FS laser or the Hansatome microkeratome (Bausch and Lomb, Rochester, NY) or no surgery. Sixteen eyes also had lamellar cuts with no side-cuts with the 30 KHz FS laser. Animals were euthanized and corneas processed as frozen sections or fixed for transmission electron microscopy. Frozen sections were evaluated with the TUNEL assay to detect apoptosis and immunocytochemistry for CD11b to detect mononuclear cells. Cell morphology was examined with transmission electron microscopy.

Results:: Rabbit corneas that had flaps with the 15 KHz FS had significantly more stromal cell death and greater monocyte influx in the cornea at 24 hours after surgery than corneas that had flaps with the 30 KHz FS, the 60 KHz FS or the microkeratome. The 60 KHz FS results were not significantly different from the microkeratome results for any of the measured parameters at 24 hours, except for mitotic stromal cells at the flap margin. Even though the TUNEL assay detected keratocyte apoptosis at 24 hours after formation of FS flaps, transmission electron microscopy revealed that the primary mode of stromal cell death at 24 hours after FS ablation was necrosis. This contrasts with almost 100% keratocyte apoptosis at 24 hours after flaps are generated with the microkeratome.

Conclusions:: The 15 KHz FS that utilizes higher energy levels and greater total energy for lamellar-cuts and side-cuts) triggered much greater stromal cell death and inflammatory responses than either the 30 KHz or 60 KHz FS or the microkeratome. Stromal cell necrosis associated with FS flap formation, in contrast to apoptosis that predominates after microkeratome flap formation, triggers greater inflammation after LASIK performed with the 15 KHz FS, especially with higher energy levels that result in proportionally greater keratocyte cell death. The TUNEL assay may detect necrotic cells under some circumstances and, therefore, the mode of cell death must be confirmed with other methods whenever this assay is used in a new system.

Keywords: apoptosis/cell death • cornea: stroma and keratocytes • inflammation