STANDARDIZATION OF THE MEASUREMENT AND IMAGING OF BLOOD PERFUSION OF THE SKIN MICROCIRCULATION IN TOWNES SICKLE CELL DISEASE MICE, USING THE LASER SPECKLE CONTRAST IMAGING TECHNIQUE

Abstract

Sickle cell disease (SCD) is caused by the production of abnormal hemoglobin S, whose polymerization (induced by deoxygenation) results in sickling of erythrocytes and numerous pathophysiological consequences, including painful vaso-occlusive episodes. At present, there are few non-invasive methods to detect the occurrence of vaso-occlusion (VO) in the microcirculation of pre-clinical experimental models. One alternative may be the use of laser speckle contrast imaging (LSCI) to detect changes in the microcirculation pattern of the skin. For this method, an infrared laser is positioned over a region of skin and the backscattered light can be quantified to determine the blood perfusion. Therefore, the first aim of this study was to standardize a novel non-invasive technique and the best region to quantify and compare skin perfusion in mice with SCD. Secondly, we aimed to look at the effect of administering a known vaso-occlusive stimulus (tumor necrosis factor-α; TNF-α) on skin perfusion in the Townes mice. For this, male Townes mice, aged 20 weeks and weighing ±25 g were used for determining total and regional skin microcirculation using the PeriCam PSI HR system. Mice were randomly distributed into 2 groups, according to stimuli: Saline (200 μL, i.p., n = 5) or TNF-α (0.5 μg in 200 μL, i.p., n = 5). At 3 hours post saline or TNF-α administration, the animals were anesthetized, the trichotomy of the abdominal and pelvic region was performed and animals were submitted to LSCI of the abdomen/pelvis (arbitrary units). Five different areas of skin were analyzed to standardize the best region to detect the occurrence of VO: upper abdomen (Region 1); lower abdomen (Region 2); central region of pelvis (Region 3); pelvis/hind limb (Region 4); abdomen + pelvis (Region 5). The data from the respective regions were submitted to unpaired t test (GraphPad Prism 8 software). All animal procedures were carried out with approval of the local Commission for Ethics in Animal Experimentation (University of Campinas; protocols 5790-1/6078-1). Different areas of the ventral region of mice were chosen for the analysis due to variations in vascularity and skin thickness when compared to the dorsum. Analyses of the entire images (total shaved area) of the mice demonstrated a significant reduction in the perfusion of the skin microvasculature after TNF-α (88 ± 5 units) administration, when compared with saline-treated mice (110 ±15 units) (p < 0.035). The comparison of each region in the Saline- or TNF-α-treated mice suggested that Region 5 (abdomen and pelvis) was the only region that demonstrated decreased blood perfusion (Saline: 128 ±8 units, TNF-α: 104 ± 5 units, p < 0.0335); the other regions presented reduced blood perfusion, but there was no statistical significance. Results suggest that the total area of the abdomen and pelvis is the best region to detect the variation in blood perfusion in the skin microvasculature using the LSCI technique in the Townes SCD mice model. Furthermore, the administration of a potent inflammatory molecule, known to induce vaso-occlusive-like processes in SCD mice, significantly decreased skin perfusion, confirming this method as suitable for analyzing and visualizing microcirculatory skin vaso-occlusion.