Serial Morphological Investigations Of Endothelial Cells In Human Hemangioma Xenograft Model On Nude Mice

Background and objectivesInfantile hemangioma (often abbreviated as hemangioma) is the most common pediatric tumor characterized by rapid growth in early life and subsequent spontaneous regression in the following years. Though most hemangiomas end in automatic involution, the early rapid growth of some hemangiomas with special location may compromise the function of important organs, or are life-threatening, or bring cosmetic problem and warrant medical intervention. Unfortunately, with poverty in knowledge of pathogenesis of hemangioma, current therapeutic modalities are not satisfactory in many of these cases. One of the major obstacles to clarifying the pathogenesis of hemangioma lies in scarcity of proper models which are able to reflect a more natural and benign development of human hemangioma. On the basis of previous experiences (in literature) of establishing xenograft models of human malignancies and benign tissues on immunodeficient animals, it was attempted to establish an animal model of human hemangioma on nude mice in this study, by xenografting human hemangioma tissue into nude mice subcutaneously. The grafts were observed and their morphological alterations investigated.MethodsThe original hemangioma specimen was obtained from a male infant aged 2 months by surgery. It was cut into small pieces 5mm X 4mm X 3mm in size. The pieces were grafted into sixteen juvenile nude mice subcutaneously, 6 pieces each.1. The grafts were observed on volume, color, and texture at 1, 2, 3, 5, and 7 days, and twice a week during the subsequent 12 weeks, and once a week later till the end of the 6th month.2. Grafts were harvested at 2, 3, 5, 7, 15, 30, and 45 days, and 2, 3, 4, 5, and 6 months after grafting, respectively. Formalin-fixed and paraffin-embedded specimens were cut into 6 a m sectioins and stained with HE for microscopic observation.3. Double immunofluorescent labeling, in which mouse anti-human CD31 monoclonal antibody were employed in indirect immunofluorescent labeling and FITC-labeled rat anti-mouse CD34 monoclonal antibody were employed in direct immunofluorescent labeling, was carried out on specimens harvested at different time points. Under confocal microscopy, distribution pattern and related chronological alterations of human and mouse endothelial cells were observed.4. Grafts were harvested at 2, 5, 30, and 45 days and 2, 3, 4, 6 months after grafting. Glutaraldehyde-fixed specimens were prepared into ultrathin sections for ultrastructural observation under transmission electron microscopy.Results1. During the early stage after grafting, change in volume of grafts was not obvious. Two to three days after grafting, 21 in 96 grafts turned purple with hematoma appearance when exposed in dissection. At 3 days after grafting, tiny fibrous capsule with capillary network surrounding the grafts was recognizable. Fifteen to thirty days after grafting, 13 grafts were found decreasing in volume and turning pallor, and they vanished successively before the end of 2nd month. Most of the grafts kept there volume 3 to 4 weeks after grafting, and subsequently grew to achieve the apex of volume curvature at the end of 2nd month, when they appeared purple outside and fresh red in dissection. After 3 months of growth in host, most of the grafts were in regressing and turned pallor or slightly yellow, with fibrofatty appearance in dissection. At the end of the 6th month, the volume of grafts was a third of that of original ones. In two grafts harvested at 7 and 15 days after grafting, respectively, no hemangioma-like areas were found and they were taken on as ischemic necrosis.2. Under light microscopy observation, the original human specimen of hemangioma was consisted of plump cells crowded in lobes. Small and irregular lumens containing blood cells were recognizable among the cells. Cellular edematous signs were found at 2 days after grafting. At 3 days (after grafting), fibrous capsule and subcapsular hemorrhage were found, in the central areas, cells dispersed and interstitial tissue in rarefaction. Large scale of hyaline degeneration with scattered cell groups could be seen at 5 days. Coagulation necrosis was definite in sections from specimens obtained at 15days, and the cellular structures were retained mainly in peripheral areas. At 30 days, cell groups grew in number and then grew in cellular density as evidenced by histological examination on specimens harvested at 45 days. Karyokinesis was frequent and the grafts resembled typical proliferative hemangioma in morphology. At the end of the 2nd month, the majority of introlesional area was held by plump cells among which small to slightly distended vascular lumens were identifiable. Three months after grafting, the histological structure of the grafts was sponge-like, with dramatically distended lumens and intratrabecular dense cellular clusters. Four months after grafting, the cells accumulated in lobes again with obvious interlobular fibrous bands, followed by progressive shrinkage of cell number, growth of fibrous bands and deposition of adipose. The grafts were mostly substituted by fibrofatty tissue with residual distended microvascular lumens at the end of the 6th month.3. In double immunofluorescent labeling, human endotheliocytes appeared red and mouse endothelialcytes appeared green. Plump human endothelial cells densely accumulated in nests in original human specimen. AT 3 days, human endothelial cells arrange in nests or trabecules. Between 5 to 15 days after grafting, human endothelial cells decreased in number and dispersed in distribution. At the end of the 1st month, human endothelial cells grew in number, and got the highest cellular density at 45 days. At the end of the 2nd month, human endothelial cells occupied the majority of the grafts. Slightly distended microvessels lining with flat human endothelial cells were seen then. At 3 months, sponge-like structure was constructed in grafts byhuman endothelial cells, with greatly distended lumen lined with flat cells and densely accumulated intratrabecular cell clusters. After that, human endothelial cells decreased in number and dispersed again. During the observation course, microvessels composed of mice endothelial cells were mainly distributed in peripheral area of grafts and rarely found in central area. 4. Under transmission electron microscopy observation, the original human hemangioma tissue was mostly composed of plump and irregular endothelial cells with blood vessels surrounded by one or more endothelial cells. The lumens were narrow and irregular. At 2 days, extension of mitochondria with reduced crests, and extension of endoplasmic reticula were significant. Within endothelial cells, some perfectly round vacuoles appeared in specimens harvested during 5 to 15 days after grafting. Deposition of collagen and necrosis of cells were obvious. At 30 days, endothelial cells accumulated in groups again. The cells were plumb with uniform caryoplasm and normal organelles. At 45 days, the endothelial cells accumulated densely. The nuclear/plasm ratio was high and the caryoplasm was uniform. Vascular lumens were narrow and irregular. At the end of the 2nd month, morphology of endothelial cells varied, most were plump and some were flat, especially those lining the distended lumens. Introvascular thrombogenesis and organization was frequent. The number and density of endothelial cells reduced progressively subsequently with obvious cellular necrosis, collagen deposition, and adipose accumulation. Some residual distended microvessel lined with flat endothelial cells could be found dispersely distributed within the grafts. Some plump endothelial cell groups were still recognizable at theend of the 6th month.ConclusionsIn situ animal model of human hemangioma has been established grafting human hemangioma tissue into immunodeficient nude mi subcutaneously. Most of the grafts were living. After a period of ischem the grafts grow rapidly and followed by automatical and gradual regressic It was shown in morphological investigations that the chronologic development of grafts resembled that observed in human hemangioir During the process of investigation, the vast majority of endothelial cells grafts were of human origin, indicating the grafts were somewhat intact in ti host body. This model in a way resembles the natural development of hum; hemangioma which proliferates rapidly in the early life and then involut spontaneously. Simple in operating, high in harvesting, short in investigate cycle, and remaining of in situ environment, are also priorities worthy recommending.