Laboratory Study Of Focused Ultrasound On Guinea Pig Model Of Chronic Eczema

BackgroundChronic eczema is a common disease which represent chronic, relapsing and pruritus dermatosis. However, there is no satisfactory method to treat it at present. As a recently developed noninvasive technology, high intensity focused ultrasound therapy has been used to treat benign and malignant tumors. The focused ultrasound have been used to treat white lesions of vulva, which has an enheartening therapeutic effect. There are a lot of similar places between chronic eczema and white lesions of vulva in pathogenesis, clinical situation and pathology. Whether treating chronic eczema with focused ultrasound or not has become one of considerable research directions in the focused ultrasound therapy for dermatogic diseases.ObjectTo observe the effect and safety of focused ultrasound on guinea pig model of chronic eczema, especially to study apoptosis expression and its mechanism in lymphocytes, and changes of cytokine IL-4,IL-12,IFN-γand NGF,SP in the skin of guinea pig after focused ultrasound irradiation. To investigate the possibility and mechanism in treating chronic eczema by ultrasound and to provide reference for clinical use.MethodsThe study involves 5 parts.1. 20 guinea pigs were divided into 4 equal groups, which were irradiated on the skin of the back by 1,2,3,and 4W respectively and observed pathologic and exterior changes of irradiation areas, so as to investigate a better scanning dose on guinea pigs.2. A total of 40 guinea pigs were shaved at two sites on the back. The eczema models were prepared by using 2,4-dinitrochlorobenzene as an antigen to stimulate the shaved sites repeatedly and divided into 1W group and 2W group at random. The focused ultrasound treated one shaved site but not the other shaved site. The effect of focused ultrasound on guinea pig model of chronic eczema was evaluated by the treatment scores and pathological changes after a week.3. 1W group and 2W group were observed at 24h and 48h after ultrasound irradiation. TUNEL was used to detect the distribution of apoptosis cells. The immunohistochemical method was to used to detect the distribution of Bcl-2 and Bax protein in the skin .4. 40 guinea pigs were divided into 2 equal groups. 20 guinea pigs were prepared by using 2,4-dinitrochlorobenzene repeatedly and treated by 2W focused ultrasound irradiation. The other 20 guinea pigs were normal control group. Samples were taken before ultrasound irradiation and on 14 days after irradiation. The immunohistochemical methold was used to detect the changes of cytokine IL-4,IL-12, and IFN-γprotein in the skin of guinea pigs.5. Samples of the normal control group and the eczema model group were taken before ultrasound irradiation and on 14 days after irradiation. The immunohistochemical model was used to detect the changes of NGF and SP protein in the skin of guinea pigs.Results1. Hyperemia and edema were evident after ultrasound irradiation as irradiation dose increased. Skin temperature was higher than normality. When the skin received 3W and 4W irradiation, there were lots of blisters appeared immediately on the guinea pigs skin, red thick crusts formed 6 days later, some scars without hairs shown 15 days later. When the dose were 1W and 2W, there were some blisters appeared immediately on the guinea pig skin, a small quantity of crusts and scales formed 6 days later, dense hairs without scars shown 15 days later. Microscopic changes: spongiosis and blisters formed in epidermis, hyperemia and edema existed in papillary layer of dermis. Hyperemia and edema disappeared 6 days later, chronic inflammatory cells may be observed in dermis and epidermis became thicker than normal skin.The granulation tissue and thick crusts can be observed in the skin received 3W and 4W irradiation. The hair follicles decreased and collagen fibers increased in epidermis of 3W and 4W groups 15 days later. The skin of 2W and 1W groups resumed normally 15 days later.2. The skin of morphology and histopathology of eczema models of guinea pig showed the eczematous features. After a week, a significant difference could be observed between the treatment groups and the control groups(p<0.05). The therapeutic effect of 2W group was better than the effect of 1W group. Distinct microscopic changes were presented between the treatment group and the control group. The acanthosis, spongiosis in epidermis, lymphocyte and histiocytic infiltration in dermis, hyperemia and edema in dermis of the treatment group were all improved 7 days later.3. Both the average percentages of lymphocyte apoptosis cells and Bax protein were higher in the treatment groups than that in the control groups(p<0.001).The significant difference in Bcl-2 protein between the two groups was also abserved(p<0.001). There was a positive correlated signification between apoptosis cells and Bax, and a negative correlation between apoptosis cells and Bcl-2.4. Both expression of IL-4,IL-12 and IFN-γprotein in the epidermis were higher distinctly in the model group than in the normal group(p<0.01). On 14 days after ultrasound irradiation the express of IL-4 protein was decreased, however, IL-12, and IFN-γprotein were increased than before irradiation. The significant difference between the treatment group and the control group was also observed in the expression of IL-4,IL-12 and IFN-γprotein (p<0.05).5. Both NGF and SP protein in the epidermis were higher distinctly in the model group than in the normal group(p<0.01). On 14 days after ultrasound irradiation the significant difference between the treatment group and the control group was also observed in the expression of NGF and SP protein (p<0.001). There was a positive correlated signification between NGF and SP(p<0.05).Conclusions1. The ultrasound dose of 3W and 4W could make the irreversible damage to the skin of guinea pigs. But scanning dose of 1W and 2W can insure the efficacy and safe of energy deposition.2. Guinea pig model of chronic eczema can be treated with focused ultrasound effectively and safely. Under the safe dose of irradiation the higher dose may get the better effect.3. Focused ultrasound can significantly induce the lymphocytes of guinea pig model of chronic eczema to apoptosis and modulate the expression of the apoptosis related genes Bcl-2 and Bax.4. The unbalance was presented between TH1 cells and TH2 cells of guinea pig model of chronic eczema. The expression of IL-4 protain played an important role in acute skin lesion, but IL-12 and IFN-γprotain did in chronic skin lesion. Focused ultrasound therapy can effectively keep the balance of the expression of cytokine in epidermis.5. NGF and SP play an important role in the pathogenesis of chronic eczema. Controlling the secretion and expression of NGF and SP may be one of the mechanisms of focused ultrasound treating the chronic eczema.