The influence of Multiwave Locked System (MLS) laser therapy on clinical features, microcirculatory abnormalities and selected modulators of angiogenesis in patients with Raynaud’s phenomenon

Seventy-eight outpatients with RP (75 women and 3 men; mean age 42.8 years, range 19–77) of the Department of Rehabilitation of the Medical University of Bialystok, Bialystok, Poland, were recruited into the study. All RP patients were classified into two groups, those with primary RP, 38 cases (including 36 women and 2 men; mean age 36.5 years, range 19–77), fulfilling the criteria of LeRoy and Medsger [22], exhibited exclusively Raynaud’s phenomenon without any clinical or laboratory signs of the presence of a systemic autoimmune disease and 40 patients with secondary RP (39 women and 1 man; mean age 48.9 years, range 26–66). Patients with primary RP had absence of NVC scleroderma pattern and negative findings of autoantibody assays [22]. The group of secondary RP included 28 patients with systemic sclerosis (SSc), six patients with systemic lupus erythematosus (SLE) and six cases with undifferentiated connective tissue disease (UCTD), according to the international criteria [23‐26]. Patients with the history of previous contact with vasodilators or immunosuppressive therapy with cyclosporine or cyclophosphamide, which could influence on the microvasculature, were excluded [27]. The characteristics of the patients’ groups are shown in Table 1.

Patients had not been treated with drugs which have effects on vascular remodelling and immunosuppressive agents. Patients were treated, when required, with diuretics (eight persons), anti-malarial drugs (six patients) and no more than 5 mg/day prednisone equivalent (seven patients). The control group consisted of 30 healthy volunteers, matched for sex and age, with no signs of RP.

The Ethical Committee of the Medical University of Bialystok, Bialystok, Poland, approved the study, and informed consent was obtained from all participants before entry to the examination.

Clinical, capillaroscopy and laboratory investigations were performed on the days of blood sample collection, i.e. 1 week before the start of treatment and 1 week after the last session (4 weeks after the first session) of the MLS laser therapy. The data were collected only during the winter months.

One week before the start of treatment, during the 3 weeks of the MLS laser therapy, and for 1 week after the end of exposure sessions, patients were instructed to record any Raynaud’s attacks in diaries. Number of attacks, the mean duration of Raynaud’s attack and severity of pain, assessed by means of a 10-cm visual analogue scale (VAS), had to be recorded daily at bed time.

Serum samples were frozen at −80 °C immediately after collection. Examinations included pulmonary and renal function tests, chest X-ray, renal sonography as well as erythrocyte sedimentation rate (ESR) and total antinuclear antibodies (ANA). ANA were detected by indirect immunofluorescence on human Hep-2 cell substrate (Viro-Immun Labor Diagnostica GmbH, Germany) and considered as positive when a dilution higher than that of 1:80 was obtained. The serum concentration of VEGF and Ang-2 were determined using an ELISA kit (Quantikine, R&D Systems). Assays were performed strictly according to the manufacturers’ instructions.

NVC was performed in all patients and healthy volunteers using a stereomicroscope SZ 4045 with a final magnification of ×200 (Olympus, Germany). A fibre optic light source and filter provided cold illumination. The optical microscope was connected to a colour digital camera and a personal computer with a high-resolution colour monitor (14 in.) (Olympus, Germany). Four consecutive fields extending over 1 mm in the middle of the nailfold were studied per finger using image analysis software (Imaging Software Cell*, Olympus Soft Imaging Solutions). The images taken at the time of examination were analyzed by the same experienced investigator (AKM) without knowledge of the patient’s clinical diagnosis. The investigator was blinded also referring to the capillary images recorded as a pre- and post-treatment with MLS laser therapy.

Each patient was acclimatized for 20 min at room temperature of 20–24 °C prior to the examination. The nailfolds of all fingers except the thumbs and fingers affected by recent local trauma were examined in each patient. To obtain the best visibility of the microvasculature, a drop of immersion oil was placed on the nailfold bed. Only the capillaries in the distal row of the nailfold were analyzed.

The intensity of the morphological changes was evaluated on the basis of semiquantitative method which included the parameters: loop density; capillary length variability; percent of loops with architectural derangement such as tortuous, meandering, enlarged/giant, ramified or bushy capillaries; irregular distribution of the capillary array; and the presence of extravasations into perivascular tissue. All parameters were defined on the basis of previous classifications [9, 11] as follows: dilated capillaries, microvessels with a diameter of the arterial limb wider than 0.015 mm (=15 μm) or a venous limb wider than 0.020 mm (=20 μm); giant capillary loops, homogenously enlarged loops with a diameter wider than 0.050 mm (=50 μm); elongated capillaries, microvessels with length greater than 0.500 mm (=500 μm); microhemorrhage, presence of a dark red mass characterized by hemosiderin deposit derived from capillary injury; tortuosity, a variation of the typical harpin capillary shape; and avascular area, a distance between two adjacent capillary loops longer than 0.500 mm (=500 μm). The capillaroscopic findings were graded according to previous studies [28‐30] with modifications ranging from 0 to 3 where score 0 indicated normal feature (>8 capillaries/mm, typical hairpin-shaped loops arranged in parallel rows); 1, mild (6–8 capillaries/mm, <33 % changed capillary loops with non-homogenous distribution or size of loops, elongation of the loop or shortened loops and the absence of haemorrhages); 2, moderate (33–66 % morphologically changed capillaries, enlarged loops, diminished loop density and microhaemorrhages); and 3, severe changes (more than 66 % dilated capillaries with heterogeneous features of angiogenesis with a variable capillaroscopic pattern, avascular areas and microhaemorrhages). The mean score for each subject was obtained from the analysis of all fingers.

All patients with RP received two-wave MLS laser (ASA Company) irradiation (808 and 905 nm) 5 days per week during the period of 3 weeks, with the following parameters: power 3.3 W (3,300 mW), frequency 1,500 Hz, energy 129.3 J, energy density 1.6 J/cm² and procedure time 2.5 min on single hand. The laser scanner covered the fingertips and the dorsal parts of both hands.