Differences in the ablation of the Great Saphenous Vein by 810nm and 1470nm Endovenous Lasers, as shown by histology and immunohistochemistry using a novel method of in-vitro treatment Henry F. Ashpitel1, F. Javier Salguero1, Waldo García-Jiménez1, Roberto M. La Ragione1 and Mark S. Whiteley1,2 1Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH. 2The Whiteley Clinic, 1 Stirling House, Stirling Road, Guildford, Surrey, GU2 7RF.

Disclosures & Ethics Henry Ashpitel is funded for his PhD by an educational grant from AngioDynamics. This work is a part of his PhD studies. This study received favourable ethical approval from: NHS HRA, NRES Committee South East Coast-Surrey: 13/LO/0058 University of Surrey Ethics Committee: EC/2013/23/FHMS

Introduction NICE CG168 (July 2013): Treatment of varicose veins preferentially: “Endothermal ablation”

Introduction EVLA Initially chromophore used haemoglobin 810 nm, 940 nm, 941 nm, 1064 nm More recently chromophore used water 1470 nm, 1920 nm Both 810 nm and 1470 nm commonly used Both reported to be able to ablate GSV at 40 – 100 J/cm

Aims Much clinical research into recurrence rates and patient satisfaction Little research into biological effects of EVLA on GSV To investigate: The difference in the biological effect of 810 nm and 1470 nm EVL in GSV Examine the expression of various apoptotic-linked proteins in GSV treated with the EVL’s Develop an original model of treating ex-vivo GSV

Method Ten lengths of ex-vivo extra-fascial GSV: Post-collection, tissue cultured overnight at 37˚C Five treated with 810 nm EVL and five treated with 1470 nm EVL (AngioDynamics) Each length treated in five sections: 0 (CTL), 20, 40, 60 and 80 J/cm Post-treatment tissue cultured at 37˚C. Sub-sections from each treatment group retrieved at 6 and 24 hours Sub-sections taken for histological and immunohistochemical analysis Histological Analysis: Haematoxylin and Eosin (H&E) and Martius Scarlet Blue (MSB) Immunohistochemical Analysis: anti-smooth muscle actin (αSMA) and anti-caspase-3 (αC3)

Results - Histology MSB Staining Blue – Collagen Red – Fibrin 810 nm 40 J/cm 1470 nm 40 J/cm Control 810 nm 60 J/cm 1470 nm 60 J/cm

Results - Immunohistochemistry αSMA Staining Brown/Red = Smooth Muscle Actin 810 nm 40 J/cm 1470 nm 40 J/cm 810 nm 60 J/cm 1470 nm 60 J/cm Control

Results - Immunohistochemistry αC3 Staining Brown/Red = Cleaved Caspase-3 Arrows indicate Caspase-3 expression 810 nm 40 J/cm 1470 nm 40 J/cm 810 nm 60 J/cm 1470 nm 60 J/cm Control

Analysis of Results Clear that there is a significant difference between treatment with 810 nm and 1470 nm EVL’s Significant increase of thermal damage and spread with the 1470 nm EVL at 40, 60 and 80 J/cm Confirmed with αSMA immunohistochemical staining More caspase-3 expression seen with the 810 nm EVL Very little caspase-3 expression seen with the 1470 nm EVL at 6 hours and nearly none at 24 hours Significantly less caspase-3 expression in sections treated with the 1470 nm EVL at 40, 60 and 80 J/cm compared to the control

Conclusions Thermal damage and spread increased when using the 1470 nm EVL Caspase-3 immunohistochemistry suggests: That most of the cells in tissue treated with 1470nm EVL have undergone apoptosis by 24 hours That most of the cells in tissue treated with 810 nm EVL are undergoing apoptosis by 24 hours Hence, there is a clear superiority in damage and cellular apoptosis in GSV treated with 1470 nm EVL Novel model appears to be successful and easy to use Feedback confirmed methodology simplicity and practicality