3B). Open in a separate window Figure 3. Histopathological evaluation of mouse skin samples (H&E staining). inflammation and the disease severity were assessed using measurements (erythema, desquamation and induration parameters, PASI modified score), splenomegaly assessment and histopathological evaluation. Phenotypic characterization of NK cells in imiquimod (IMQ)-treated mice was performed by flow cytometry, for both PB and spleen cell suspension. A large panel of surface markers was used: maturation and activation markers [cluster of differentiation (CD)49b, CD11b, CD43, CD27, KLRG1, CD335, CD69, CD28, gp49R, CD45R, CD11c] and markers for cytokine receptors (CD25, CD122, CD132). Our experimental data showed important differences in IMQ-treated mouse NK cell phenotype as compared to control group. The maturation markers (CD11b, CD43, CD27, KLRG1) were found increased on NK cells, in periphery and spleen, while CD49b+NK1.1+ was significantly lower, and the alterations correlated with the severity of the disease. Our findings reflect the immune engagement toward activatory profile of NK cells and draw attention to evaluating Ps intensity correlated with the mature profile of circulating NK cells. (29). AZD5423 In order to aid information to the pathogenesis of Ps and to identify potential therapeutic targets, numerous experimental models performed and/or were developed, each of them heaving advantages and disadvantages. mouse models of Ps can be grouped into spontaneous (chronic proliferative dermatitis cpdm/cpdm, flaky skin measurements AZD5423 (erythema, desquamation and AZD5423 induration parameters, PASI modified score), splenomegaly assessment and histopathological evaluation. In vivo measurements of skin inflammatory parameters Erythema, desquamation and induration parameters (EDI) were assessed to study the progress of skin inflammation and hence the disease severity by daily monitoring. EDI were scored daily on a scale from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; and 4, very marked. Fig. 1 presents a representative case of inflammation induced by IMQ and the EDI scoring for all the mice in the IMQ group matched the pattern previously published by us for this experimental model (34). As previously NR4A3 shown, all EDI parameters, erythema is the first parameter that can be scored after one day of IMQ applications, followed after another day by the subsequent registered parameters, thus starting from day 2, all EDI parameters are registered in all the animals subjected to IMQ (34). Open in a separate window Figure 1. The evolution of back skin inflammation during the IMQ-treatment. On day 1, the IMQ-based treatment was initiated, and on day 6 AZD5423 the mice were sacrificed. IMQ, imiquimod. As in psoriatic patients, in our animal model, the severity of inflammation was estimated based on a modified PASI score (0C12 scale), calculated daily by adding the independent daily scores obtained for EDI (the affected area was not taken into account). The PASI score had a progressive evolution during the IMQ-treatment (Fig. 2) matching the increased severity of the psoriatic lesions. Open in a separate window Figure 2. measurements of PASI cumulative score. PASI cumulative score was calculated daily by adding the scores obtained for erythema, skin scaling and thickening: D1: 0; D2: 1.30.5; D3: 50.9; D4: 6.70.8; D5: 9.71; D6: 10.21.2. The results are presented as mean score SD; n=6. n, number of mice; D, day; PASI, psoriasis area severity index. As the PASI score clearly depicted the evolution of the psoriatic lesions we evaluated the histopathology of psoriatic-like skin in our model. Histopathological evaluation After 5 days of treatment, IMQ-based cream induced pathological alterations in the epidermis, by compromising its integrity. Several histopathological features that are typical for human Ps, such as hyperkeratosis, parakeratosis, acanthosis and elongation of rete ridges were observed (Fig. 3B). Open in a separate window Figure 3. Histopathological evaluation of mouse skin samples (H&E staining). (A) Normal skin. (B) IMQ-treated skin. IMQ treatment induced hyperkeratosis, parakeratosis, acanthosis and elongation of rete ridges (B). IMQ, imiquimod; H&E, hematoxylin and eosin. Splenomegaly assessment At the end of the experiment (day 6) the mice were weighed and sacrificed; spleens from all the animals were removed and weighed in order to assess the splenomegaly, SW/BW ratio (spleen weight/total body weight) was calculated. The IMQ-treated mice SW was significantly higher compared to healthy mice (control group) (0.2150.03 vs. 0.090.02, P=4.910?4) (representative measurement is presented in Figs. 4 and ?and5A).5A). When assessing SW/BW ratio we found that in IMQ-treated mice this ratio was 2.5 times greater compared to control mice.