Sir,

The release of tumor necrosis factor-α (TNF-α) has been reported to be increased in human tear fluid during the first 2 postoperative days following excimer laser phototherapeutic keratectomy (PTK).1 Interleukin-6 (IL-6) has also been found to be increased in the human tear fluid from 12 PTK patients, as measured 24 h after PTK.2 In this study we have analyzed the gene expression of TNF-α and IL-6 in rat corneas after PTK.

In all, nine eyes of nine Wistar rats received excimer laser PTKs (B&L Kerakor 217 laser (Bausch and Lomb, Chiron Technolas GmbH, Dornach, Germany), optical zone 4 mm, 1600 pulses, nominal ablated depth 50 μm). Three groups of three rats each were killed at 1, 12, and 24 h after treatment, respectively. An additional group of three rats without previous PTK served as control group. From all the collected eyes, 4 to 5 μm paraffin sections were obtained on RNAase-free silan-coated slides and further analyzed by nonradioactive mRNA in situ hybridizations, using the DIG-labeling and detection kit from Roche Diagnostics (Mannheim, Germany), as described.3 Statistical analysis was performed using the two-tailed Mann–Whitney U-test, and differences were considered significant at P<0.05.

At 1 h after PTK, the gene expression of the cytokines TNF-α and IL-6 was higher than in untreated controls, but lower than 12 h after treatment (Table 1). The increases observed between 1 and 12 h after PTK were statistically significant for both cytokines (P=0.0005 and P=0.0078, respectively; Table 1). The expression of the inflammatory cytokines TNF-α and IL-6 was detected not only in epithelial, endothelial, and infiltrating cells,4 but also in the keratocytes from the corneal stroma (Figure 1). Whereas at 24 h after PTK, the expression of both cytokines remained higher than in the controls, slight decreases could be observed when compared with the results at 12 h after PTK treatment (P=0.0244 and P=0.0142, respectively; Table 1).

Table 1 TNF-α and IL-6 gene expression in corneal sections from Wistar rats, as detected by specific nonradioactive in situ hybridizations, at 1, 12, and 24 h after PTKa
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Figure 1
figure 1

Detection of TNF-α (a) and IL-6 (b) gene expression using nonradioactive in situ hybridizations in paraffin corneal sections from Wistar rats at 1, 12, and 24 h after PTK. (a) Representative TNF-α mRNA hybridization pattern in a paraffin section from rat 2 of group 4 (24 h after PTK, hybridization score 3=++). (b) Representative IL-6 mRNA hybridization pattern in a paraffin section from rat 1 of group 4 (24 h after PTK, hybridization score 3=++). A positive reaction was evaluated as a dark blue–violet color, which was the result of the corresponding reaction using the DIG detection kit from Roche Diagnostics. TNF-α, TNF-α mRNA; IL-6, IL-6 mRNA. Original magnification: × 1000.

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TNF-α derived from epithelial cells after laser treatment has been found to modulate apoptosis and expression of chemokines, which would further attract monocytes and granulocytes into the corneal stroma.5 Moreover, IL-6 released from epithelial cells and keratocytes after PTK has been reported to modulate the synthesis of collagen and the production of metalloproteinases.2 All these effects may have a relevant role in wound healing after laser treatment.