Proof of new collagen synthesis in lid skin after Er:YAG laser skin resurfacing with in‐situ hybridization

Abstract

Abstract Purpose: The aim of the study is to prove new collagen synthesis in lid skin fibroblasts and correlate it to laser energy fluence after Er:YAG laser skin resurfacing, using the method of in situ hybridization. Methods: Lid skin of 9 patients with blepharochalasis was treated with laser at day 7 and 21 before elective blepharoplasty, when it was excised for further investigation. We used a 2940 nm Er:YAG laser (Fidelis by Fotona) with smooth mode parameters: laser fluence 0.75 – 2.00 J/cm2, spot size 5 mm, repetition rate 20 Hz, pulse length 550 μs, 6 pulses per packet, packet length 250 ms, no overlapping. We designed 2 oligonucleotide RNA probes for pro‐alpha 1 and pro‐alpha 2 collagen and labeled them with radioactive Sulphur 35. At time of surgery the excised tissue was frozen in dry ice. Tissue sections were incubated with the probes and marked with film emulsion for 10 days. We counted silver grains over specific cells, which were identified with imunohistochemistry. Results: We noticed significant elevation of pro‐alpha‐collagen mRNA expression in the laser treated areas of lid skin compared to untreated areas. Pro‐alpha‐collagen mRNA expression was particularly dense in cells, identified as fibroblasts by imunohistochemistry. Pro‐alpha‐collagen mRNA expression in these cells correlated with laser fluence used in treatment of lid skin. Conclusions: The method of in situ hybridization brings a definitive proof of new collagen synthesis in skin fibroblasts after Er:YAG laser skin resurfacing; and it is possible to measure the rate of new collagen synthesis as a function of the applied laser fluence.