| Destination:The study aimed to investigate the changes of histology and ultrastructure of melanocytes and melanin in rabbits,skin after superficial hypothermic cryotherapy with liquid nitrogen,hoping to provide the foundational theoretical support for the treatment of depigmentative diseases,such as vitiligo and so on.Methods:1,The superficial hypothermic cryotherapeutic experiment was carried out by using cotton swabs dipped in liquid nitrogen in experimental group,the liquid nitrogen is scraped on the skin of back and haunch of rabbits softly and repeatedly(about 1s) so that a thin layer of frost was emerged,unti the frost melted naturally,three times for a freeze-thaw cycle.the superficial hypothermal cryotherapy was carried out again after one week, a total of 3 cycles.The another group and the area of self-control were not freezed as control .2,To observe the change of cutaneous tissue after superficial hypothermic cryotherapy with liquid nitrogen,mainly the chang of pigmentation and the growth of hair and so on.3,The cutaneous tissues which were symboled by gentian violet were incised in the experimental and control groups separately in the third and fifth week, the HE staining and silver nitrate staining of Masson-Fontana were carried out .individually, The number of melanocytes in the stratum basale of the epidermis and outer root-sheath of hair follicles ,hair follicles and dermal capillaries were observed. Analyzed and photographed by using Mias pathological workstation 4.0 system. Meanwhile ,the specimens were prepared to electron microscopy sections strictly by the instructions, the change of morphology of melanocyte and melanin was observed,analyzed and photographed in the experimental and control groups separately by TEM.4,Statistical analysis: the data of the third and fifth week was analyzed by paired-samples t test in SPSS13.0 respectively.Results: 1. Macroscopic observation:Pitchy pigmentation gradually appeared in some experimental regions after the second week,meanwhile,some gray-black and thicker rabbit hairs erupted compared with the control group. In the fifth week pigmentation gradually spreaded all over the regions of cryotherapy ,the new rabbit hairs close to nomal length.At the same time there were two small hyperpigmentary regions in the control group, however pigmentation did not appear in the other regions.2. HE staining and silver nitrate staining of Masson-Fontana: melanin and melanocytes were significantly increased (P <0.01) in stratum basal of epidermis and in the outer root-shooth of hair follicle,the distribution of melanin granules spreaded widelye. Many melanin granules were also seen from stratum spinosum to stratum granulosum .The upper dermal capillaries were expansive and congestive,the number of capillaries was increased significantly(P <0.01),especially around the hair follicle. The corresponding item of analyzed by paired-samples t test or revised t test, there were significant differences comparing with the control group (P<0.01).But four homologous indexs of the third and fifth week in the experimental group were not significantly different statistically(P>0.01).3. Electron microscopic section : the experimental group: nuclear notchs of melanocytes were less compared with static and mature melanocyes,there were lots of melanosomes around the cellular nucleus which were at different stages,mainlyâ…¡andâ…¢stage, the melanosomes ofâ…£stage were less relatively.In the cytoplasm mitochondria was dense comparatively,the number of rough endoplasmic reticulum Golgi's apparatus and free ribosomes was increased significantly,the lumens of mitochondria and rough endoplasmic reticulum were extensive prominently.There were lots of melanin granules in the epidermis especially from stratum spinosum to stratum basal.The control group: nuclear notchs of melanocytes were more compared with the melanocytes of the experimental group, there were a few melanosomes around the cellular nucleus which were at different stages,mainlyâ…¡stage;mitochondria,,rough endoplasmic reticulum Golgi's apparatus and free ribosomes were scattering sparsely in the cytoplasm. There were a few melanin granules in the epidermis.Some of mature melanin granule(â…¢stage) exited in the part of melanocytes closing to stratum spinosum.Conclusion:1. The superficial hypothermic cryotherapy can stimulate melanocytes in the epidermis and hair follicles, accordingly the function was enhanced significanly,more importantly the kind of dopa-negative melanocytes was activated and start to synthesis melanin abundantly, so that the melanin was inscreased significantly within the epidermis and hair follices.Meanwile newborn hairs are blacker and thicker,and grow productively.2. The superficial hypothermic cryotherapy which is safer and lower adverse is feasible in the basical theory to treat this kind of depigmentative diseases such as vitiligo and so on,this expertiment supplies theoretical support for this therapy method.
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