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• To determine the laser beam penetration profile through ex vivo skin at varying fluences from an NMRL.

• To determine the maximum depth o f penetration of photon energy through ex vivo

skin from an NMRL beam using varying fluences.

3.3 METHOD

3.3.1 Thermal Imaging Experiment

Hair-bearing scalp skin was obtained from five, consenting Caucasian patients undergoing elective face-lift procedures. Each specimen was divided into 6 pieces of approximately 12 mm^ and just prior to use had the dermal aspects microdissected to reveal the hair bulbs and the lower hair shafts. Sterile isotonic saline was regularly applied to the dermis during the whole procedure to prevent drying. For each specimen, two pieces were treated with the NMRL ("Chromos 694 dépilation", SLS Biophile, Llanelli, Wales; pulse duration 900 psec, spot size 7 mm), two acted as positive controls and two as negative controls (see below for further details). Those specimens undergoing laser irradiation were mounted on a cork ring on a sterile jig so that the epidermal aspect faced the laser, whereas the thermal imaging camera system ("Thermovision 900", FLIR Systems, Leighton Buzzard, England) was focused on the exposed hair bulbs of the dermal aspect. The dermal aspect was photographed to allow later comparison with the thermal images. The skin was then exposed to a single pulse of 15 J/cm^ (typical o f the clinical range) from the laser. Simultaneously, the thermal imaging system (frequency 15 Hertz) recorded the temperature changes from the dermal side over approximately 45 seconds, the time taken for all heat produced to dissipate. The 7 mm diameter target area to be exposed to the laser had been identified prior to irradiation by a helium-neon aiming beam incorporated into the laser system. Running the thermal imaging camera before ruby irradiation revealed that this “targeting” laser did not produce heat in the specimens. The target area was marked by pen outside the beam, so as not to interfere with the laser and was excised from the specimen by scalpel after the experiment.

Chapter 3 - The Interaction between a Laser Pulse and Human Skin

The two skin pieces from each patient that were to be used as positive controls for cellular damage (as demonstrated by expression o f p53) were exposed to ultraviolet radiation (Ultraviolet light source Nikon Ltd, model HB-10103AF, 100 Watt bulb, filtered to give wavelengths greater than/equal to 300 nm) upon their epidermal aspect for 10 minutes. These specimens were maintained throughout upon saline-damp gauze to prevent dehydration. The negative controls were exposed to air, mounted on a dry cork ring for the same time duration (1 minute and 30 seconds) as the laser irradiated specimens had been during treatment.

After the procedures, all specimens (laser irradiated and controls) were incubated at 37°C for 18 hours in culture media (Dulbecco's Modified Eagles Medium with 10% Foetal Calf Serum, 1% 1-Glutamine and 1% Penicillin and Streptomycin) with the epidermis uppermost and at the air-liquid interface to mimic the skin's natural environment. The specimens were then processed for routine paraffin wax histology and sectioned tangentially (parallel to the plane o f the skin surface) throughout their depths. Two consecutive 4 pm thick sections were taken for staining every fifth section. The first of the pair was stained by the modified SACPIC technique (Nixon, 1993) and the second by an immunohistochemical technique for the p53 protein. The modified SACPIC staining technique was initially promoted for its ability to differentiate the different growth phases of hair follicles, but has also been described elsewhere as being able to differentiate damaged from undamaged hair shafts in ruby laser irradiated specimens (see section 2.3.1) (Liew, 1999). The marker of cellular damage, p53, was detected as described in section 2.3.2. These sections were then counterstained with haematoxylin. Such alternate staining o f two consecutive sections throughout the specimens allowed comparison o f the two so establishing if a correlation existed between damaged hair shafts (demonstrated by modified SACPIC) and p53 expression within the viable cells o f that same follicle. The depth to which damage to the hair shafts and p53 expression extended in each o f the specimens was also estimated by knowing the thickness of each section (4pm) and the number of sections from the epidermis to the level where shaft damage or p53 expression was no longer present.

3.3.2 Laser/Skin Penetration Experiment

Large specimens o f normal Caucasian human skin (approximately 20 cm x 10 cm) from breast reduction procedures were taken immediately after excision from each of six patients (mean age 36; age range 28 to 44). A total o f 8 skin samples of varying thicknesses were removed from each specimen using both an air-driven dermatome for thinner samples and dissecting scissors to obtain thicker samples. The skin samples were wrapped in saline damp gauze to prevent drying and removed individually only when required during the experiment.

An external energy meter (section 2.1.6) was used to accurately measure the photon energy output from the NMRL. Since the skin samples were to be placed on a coverslip to prevent contamination of the meter, various fluences from the laser were fired directly onto the meter and compared with those noted when the laser was fired through a grade 1 coverslip, which is the thinnest available.

A second pilot study was performed to clarify whether repeated firing of the laser onto the same spot of skin would not affect the subsequent light penetration measurements. A skin sample was therefore positioned on a coverslip over the energy meter as detailed above. The laser was fired three times upon the specimen at three different fluences (4.75, 9.24 and 13.41 J/cm^) which are doses synonymous with clinical exposure, and an energy meter reading taken. This was then repeated upon a second and a third skin sample each of differing thicknesses and the results noted.

For the main experiment, the skin graft samples were removed from the saline-damp gauze in turn, trimmed to approximately 1 cm^ and the thickness measured. If the graft samples were thin then a micrometer was used taking care not to crush the tissues. If the specimen was thicker than several millimetres and contained subcutaneous fat then Wemier calipers were used to assess depth. The skin graft samples were then placed dermis down upon a coverslip taking care not to trap any air bubbles. Excess saline was removed and the samples laser treated three times at the aforementioned fluences. The subsequent energy reading was recorded.

Chapter 3 - The Interaction between a Laser Pulse and Human Skin