| BackgroundTissue defects or deep tissue of the body, such as fascia, muscles, nerves, or blood vessels, nerves, exposed by many reasons including trauma, post-tumorectomy, diabetic ulcers, gangrene, bedsores or congenital malformations ect., need to be repaired. The common way to repair the defects in clinic is to reconstruct the tissues of the body and stop the deep tissues being exposed by cutting a perforator-based flap which is composed by the tissues arrounding the perforator. The perforator-based compound tissues used as autologous transplantation donor to reconstruct tissue and prevent tissue from being exposed is known as perforator flap. In recent decades, the perforator flap is a promising new technique based on the musculocutaneous flap, with the development of the equipments of microsurgery, and is used to repair the defects, cutting tissue flap supplied by a special perforator artery without the muscles, so that it causes lesser secondary damage to donor site.Perforator flap harvests only a small donor area for the reason of cutting a small cutaneous perforator artery instead of truck artery. It is hard for surgeon to reconstruct the big defects by covering them with single axial perforator flap. Super-sized flaps based on 2 or more than 2 perforator arteries were designed to settle the defects with big size, and the key question of harvesting a super-sized flap is how to design and enlarge the flap. The stability of the blood supply and microvascular anastomose between each two perforasomes play a key role in increasing survival rate of the flap.Early in 1987, Taylor et al. made a detailed research about cutaneous artery on human and some other mammals, using technique of angiography, claiming that there were 374 cutaneous perforator vessels with an average diameter of 0.5 mm or more in human beings that could be cut off. Moreover, they put forward a concept of Vascular Territories,based on the three-dimensional characteristics of vessels distribution from deep fascia to skin. They believed that every well-known vessel in skin was not only responsible for the skin and the subcutaneous tissue of its distribution, but also supply blood for many of the tissues of the same area, such as muscles, tendons, bones, etc, which is called angiosomes. According to their search, our body can be divided into over 40 angiosomes, connected by microvascular anastomosis between the two angiosomes (that is the choke vessels). The proposal of the concept of angiosomes not only gives rise to new donor sites and new transplantation ways of flap, but promotes the improvement of traditional skin flap transplantation as well.Nearly in the same time, Cormack et al. divided the territories of blood supply of skin into three levels, anatomical territory, dynamic territory and potential territory, by analyzing the relationship between the neighbouring angiosomes. That is to say: Firstly, anatomical territory can be defined as a solid region with a tree shaped artery, which is the fundamental territory, just like angiosomes put forward by Taylor. Secondly, every anatomical territory has abundant anastomose between neighbouring arteries. When one side of the vessel is cut off or blocked, then the blood pressure drops. At this point, blood supplying from the anatomical territory will donor the above area, with the help of choke vessels. The expansion part that is close to anatomical territory is called dynamic territory. Thirdly, if the blood can be supplied to the neighbouring area a little further, then further neighbouring area is called potential territory. It shows that it is possible to a larger flap if it is expanded from anatomical territory to neighbouring dynamic territory. An even larger flap is available if it is extended to the neighbouring potential territory. But we have to realize that, whether there exists vascular anastomose (that is choke vessels) between different territories is essential, because vascular anastomose plays a key role in turning three different small territories into a large one by establishing new blood circulation system.Therefore, the morphological features of each vascular network in each territory, and how to augment vascular anastomose has become the most important project.Earlier researches on microvascular anastomose in perforasomes were mostly performed by perfusion on cadaver. Because of the non-physiological of the cadaver, the study of the small vessels on it can't necessarily reflect the physiological state in vivo, besides, because of the factors such as the type of perfusion agent, viscosity, perfusion pressure, the effects of the perfusion can't get an ideal state. In the project, SD rats are selected to make flap model, by installing self-designed and advanced skin observation-window, we can detect the VEGF-mRNA of the obtained tissue in molecular level and summarize the proliferation law of anastomosis in perforasomes under direct vision in real-time. In addition, microvessels are perfused and marked at different time points in hope of restoring a three-dimension structure of microvascular anastomose in different state.Objective1. Improve the skin observation-window in the preliminary design, in order to design and create better observation-window that meets the demand of the experiment. Directly observe the augmentation and anastomosis of the choke vessels in rats by using skin observation-window and draw the initial rules.2. Observe the effects of MSCs-rat (bone marrow-derived mesenchymal stem cells of rat) and VEGF-rat (vascular endothelial growth factor of rat) exert on choke vessels at different times.3. Skin tissue of the anastomosed region were taken and underwent the real time PCR and immunohistochemical tests, to make sure about the change law of VEGF-mRNA.4. Sprague-Dawley rats undergo whole-body gelatin-lead oxide injection and the skin was removed for radiography to analyze the skin vasculature, then calculate the grey value as the density of capillary of the anastomosed region, analyze the value between the different groups and different time points.5. Preliminarily discuss the value of laser confocal microscope in the three-dimensional reconstruction of vessels in rats' skin, combined with Evans Blue's marking in vascular endothelial cell, in order to study the morphological features of choke vessels of neighbouring angiosomes in rats' skin and explore simple, practical, preciser three-dimensional visualized technology of vessels.MethodsThe first partThis part is the preliminary part of the project. Get a preliminary of the distribution of the main perforators in rats of back, relationship of artery and vein by the lead oxide-gelatin imaging and the video of observation area. In addition, select 12 young SD rats between 60 to 80g, extract and cultivate MSCs, in order to meet the demand of the experiment.The second and third parts are the main parts of the project.The second partSelect SD rats as experimental subject and make the flap model in SD rats by installing the self-designed, self-improved skin observation-window on the back of rats. Then the rats are divided into 3 groups randomly, which can be named as observation-window group, MSCs group and VEGF group.1. Observation using stereoscopic microscope:select 18 SD rats and divide into 3 groups randomly. Firstly, in the observation-window group, a long real-time observation into the change in the caliber and density of the microvascular anastomose (choke vessels) of the angiosomes of the skin in the back of the rats and the on-going of the delayed expansion of the microvascular anastomose by installing the skin observation-window, with the help of stereoscopic microscope. Secondly, the MSCs group and VEGF group. Apply MSCs and VEGF respectively to the two groups. A long real-time observation into the change in the caliber and density of the microvascular anastomose (choke vessels) of the angiosomes of the skin in the back of the rats and the on-going of the delayed expansion of the microvascular anastomose by installing the observation-window, with the help of stereoscopic microscope. At the same time, we explore the influence of angiogenesis related factors have on the expansion of microvascular anastomose.2. Laboratory detection:select 72 SD rats and divide into 3 groups randomly. Then take samples of the skin on the back of the observed rats in every groups according to different time points, after that, do HE stain, Rt-PCR and immunohistochemistry on the samples to detect the expression level of capillaries. Later, make a contrast of the differences between different time points and different groups, using statistics software and summarize the change law of the microvascular anastomose between angiosomes and explore the factors affecting that,The third partIn this part, there is no need to divide rats into groups or to install skin observation-window. Instead, we analyze the density of the vessels of the proliferation in microvascular anastomose according to different time points, by using perfusion and marking technique, and try to restore a three-dimensional structure of microvessels.1. Lead oxide-gelatin perfusion imaging. Select 48 SD rats and number them randomly, then with the help of.Lead oxide-gelatin perfusion imaging technology, perfuse the systemic arteries of the rats according to different time points, after that, skin from the abdomen midline of the rats, which is photographed by X-ray, and analyze the density of the anastomotic vessels and its change, using soft Scion Image Beta 4.02.2. Fluorescent tracer technique. Select 8 SD rats and label vascular endothelial cell using fluorescent tracer technique, then remove moderate epidermic cells, using decellularization technique and observe, whole-layer scan through (two-photon)laser confocal microscopy, trying to restore a three-dimensional structure of the microvascular anastomose in a physiological state in MIMICS 13.0.The main steps are as followed.The first part1. Extract, cultivate, collect, cryopreserve MSCs. Select 12 young rats weighed about 70±10g, then extract and cultivate using whole bone marrow adherent culture method, meanwhile examine cell surface antigen CD90?CD45 using flow cytometer to identify the cells and collect and cryopreserve 3rd generation MSCs.2. The distribution of angiosomes in the skin of rats. Select 6 SD rats, after anesthetizing and unhairing, perfuse the gelatin-lead oxide mixed suspension in the left ventricle, then extract the skin and photograph it by X-ray to observe the main perforasomes and the anastomosis between them, making preparation for the selection of perforasomes in the next step.3. Pinpoint the distribution of the cutaneous arteries and cutaneous veins in rats. Select 6 SD rats, after anesthetizing and unhairing according to the results of X-ray scanning, select proper perforasomes to obsereve the microvascular anastomose, which is between the left and right iliolumbar artery, install skin observation-window in rats for the purpose of venous indwelling needle through common carotid artery, then perfuse the Evans Blue solution, video and record the blood flow changes in the skin observation-window and the on-going of concomitant relationship of the arteries and veins.The second partSelect 90 SD rats, after anesthetizing, unhairing, making models of the flap and installing the skin observation-window, divide the rats into 3 groups randomly and equally, naming observation-window group, MSCs group and VEGF group.with each group 30 rats. In MSCs group and VEGF group, except for injecting MSCs and VEGF locally, other steps are the same as the steps of observation-window group.1. Observation under stereoscopic microscope. After selecting 6 SD rats randomly, from every group, make observation records by photographing of the anastomosis of the perforasomes through the observation-window under stereoscopic microscope according to different time points, they are DO(0-hour), D1 (24-hour), D2(48-hour), D3(72-hour), D4(96-hour), D6(6-day), D10(10-day) and D16(16-day) respectively. Then import the photos into Image Pro Plus 6.0 and examine the diameter of the vessels according to different time points and make a contrast of the difference of different time points and different groups.2. Molecular Biological Detection and Histochemical detection. Extract the samples of the skin tissue from the back of the rats at D0, D1, D2, D3, D4, D6, D10 and D16 respectively.24 rats from each group, that is 72 rats totally, then every time we extract samples from 3 rats in each group and two samples from every rats, that is to say 6 samples from 3 rats every time in each group. Then do the HE stain and immunohistochemistry detection on the samples acquired, according to the results of immunohistochemistry detection, do the Rt-PCR detection of the samples that have significant changes of vascular proliferation at 3 different time points to make clear of the laws of the levels expression of VEGF-mRNA of skin tissue.The third partAfter selecting SD rats and nesthetizing, unhairing, cut off the flap but remain the vascular pedicle of the perforasomes, ligeting the pedicle by silk thread, then orthotopic suture the skin (no need to install the skin observation-window), follow the laws of microcirculation augmentation that is summarized from observation-window group, remove the silk thread in different times.1. Lead oxide-gelatin perfusion of the systemic circulation through the left ventricle. Select 48 rats and after nesthetizing, do the nesthetizing and perfuse lead oxide-gelatin suspension at D0, D1, D2, D3, D4, D6, D10 and D16 respectively,6 rats at every time point then. After the gelatin become solidified, skin from the abdomen midline of the rats and photographed it by X-ray, which is later imported into Scion Image Beta 4.02, calculate the valley-peak ratio of perforasomes and the anastomosis between them and analyze the density of the vascular anastomose in perforasomes and the change law of it. (Each perforasomes has a higher density than the anastomosis between the two perforasomes, therefore, the former is grayer than the latter. In other words, the former is regarded as the peak and the latter the valley.)2. Evans Blue marking of the systemic circulation through the left ventricle. Select 8 rats. Following the laws of microvessel augmentation, perfuse and mark them at every time point. After nesthetizing the rats, lavage the systemic circulation vessels by left ventricular intubation, then inject the saline solution rapidly to fill the systemic circulation vessels and label the vascular endothelial cells, later, remove part of the epidermic cells, skin from the abdomen midline of the rats, then monitor and whole-layer scan through laser confocal microscopy(or (two-photon laser confocal microscopy), import the photos into MIMICS 13.0, trying to restore athree-dimensional structure of the microvascular anastomose in a physiological statein MIMICS 13.0. 4. Statistical treatment All the statistics of the three parts of the experiment are obtained from the SPSS13.0 Statistics Software. All the measurement data are showed as ±s. ONE-WAYANOVA is used in homo variance in each group, while Welch F is used in non-homovariance, REAPTED MEASURE ANOVA is used in measuring data repeatedly, P<0.05 means that the difference is statistically significant.Results The first part 1. Amplify in Vitro to get rats MSCs with good homogeneity and fecundity. The primary generation of MSCs-Rat are mainly spindle shaped, some triangular orpolygonal, observed by Inverted microscope, they grow adhesion to the bottom in acolony-like way with an obvious direction. The passage cells are mainly spindleshaped and with a typically radical growth. The ell morphology within fivegenerations is stable with a rapid proliferation rate. The detection from flowcytometry shows that the third generation of MSCs has specific surface markers:CD90 positive with a positive rate 96.8% and CD45 negative, positive rate was 1.2%,which suggests that the adherent third generation cells are mainly MSCs.2. With X-ray scanning after gelatin lead-oxide perfusion, we find that the main perforasomes in rats include lateral thoracic artery (LTA), thoracodorsal artery (TDA),subcostal artery (SCA), iliolumbar artery (ILA), superficial epigastric artery (SEA),superior gluteal artery (SGA). In the upper back, the anastomosis is obvious innucleus raphe both horizontally(that is between the left and right thoracodorsal arteryand the left and right subcostal artery) and vertically (between iliolumbar artery andsubcostal artery), while in the lower back, there is no obvious anastomosis with a sparse distribution of the microvessels of the anastomosis, both vertically (between iliolumbar artery and subcostal artery) and horizontally(between left iliolumbar artery and the right one). The anastomosis in horizontal nucleus raphe in the perforasomes between the left iliolumbar artery and the right one is the ideal one to observe the proliferation of micro vascular anastomose.3. By installing the skin observation-window and Evans Blue Perfusing, we can observe through the observation-window that the arteries of the angiosomes are dyed first, then it is expanded. In 20 to 30s, the arteries are stained while the veins not. It is clear to see the concomitant relationship of the arteries and veins. Furthermore, the diameter of the artery is slim and the distribution is sparse, while the vein with a thick diameter and a wide distribution. In 50s, the veins are fulfilled by Evans Blue and dyed blue.The second part1. Observing the effect of the different measurements at different time points and between different groups by integrated microscope, we can draw a conclusion that the rate of vessel proliferation at different time points is significantly different (1.14±0.14, 1.33±0.18,1.50±0.26,1.97±0.32,1.98±0.47,1.75±0.49,1.89±0.39,F=32.965, P<0.001) and the rate of vessel proliferation at different time points in each group is significantly different, too(F=11.726, P<0.001, F=19.893, P<0.001?F=6.262, P<0.001). The curve of MSCs group increase gradually and has an inclination of gradual decline after reaching the peak, while the curves of skin observation-window group and VEGF group increase gradually, when reaching the peak, it declines, but it has a inclination of ascending. The levels of vessel proliferation among 3 groups are significantly different (1.54±0.42,1.83±0.52,1.58±0.39, F=6.981,P=0.005)and there is a significant difference among D3, D6 and D10 (P=0.014, P=0.037, P=0.003). At D3, D6 and D10, the rate of vessel augmentation in observation-window group is much lower than that in MSCs group, which means that the difference is statistically significant. At D6 and D10, the rate of vessel proliferation in VEGF group is much lower than that in MSCs group, and the difference is statistically significant. The rates of vessel proliferation between observation-window group and VEGF group have no significant difference at 7 different time points. There is no interaction effect between different time points and different groups (F=2.064, P=0.059).2. HE stain.In observation-window group, we can observe the distribution of medium and small vessels, but the density of the small vessels and microvessels is relatively low, the density of D3 microvessels has a small increase. In MSCs group, a great number of small vessels and microvessels can be observed all the time, among them, D6 has the highest density. In VEGF group, small vessels and microvessels can be observed all the time, too, but it has a relatively lower density with a little increase of the density of D3 microvessels.3. Immunohistochemistry detection of the expression of endothelial cells in vertical section (marked by Factor ?). Observe the effects of different measurements that have on angiogenesis in flaps between different time points and different groups: angiogenesis between different time points has a significant difference (2.11±1.01, 3.03±1.53,2.39±1.29,2.16±1.36,1.84±1.36,1.67±1.28,1.27±0.99, F=50.695, P<0.001), angiogenesis between different time points in each group has a significant difference (F=15.751,P<0.001, F=22.610, P<0.001 and F=24.676, P<0.001). The curves of 3 groups rise gradually and have an inclination of gradual decline after reaching the peak. The levels of angiogenesis are significantly different among 3 groups (0.84±0.41,3.13±1.34,1.65±0.95, F=313.980, P<0.001). At all time points except D0, there is a significant difference among 3 groups, with P<0.001.The level of angiogenesis in observation-window group is significantly lower than MSCs group and VEGF group at all time points except D1, and the level of angiogenesis in MSCs group is significantly higher than VEGF group at all time points except D1. There is an interaction effect between different time points and different groups (F=8.733, P<0.001).4. According to the results of the detection of immunohistochemistry and integrated microscope, select D0, D3, D6 as the time points for Rt-PCR detection. The level of expression of VEGF-mRNA in skin at differnt time points and among different groups:there is a significant difference of the expression level of VEGF-mRNA at differnt time point (1.56±0.21,7.47±3.71,13.82±6.96, F=244.617, P<0.001), there is a significant difference of the expression level of VEGF-mRNA at differnt time points in every groups as well (F=118.678, P<0.001, F=98.100, P<0.001 and F=138.817, P<0.001). The curves of 3 groups have an inclination of rising. The expression level of VEGF-mRNA at D3 and D6 is greatly higher than that at D0, and the expression level of VEGF-mRNA a t D6 is significantly higher than that at D3. The expression level of VEGF-mRNA among 3 groups is significantly different (4.47±2.84,12.08±9.20,6.30±3.97, F=110.135, P<0.001), it also has a significant difference among 3 groups at D3 and D6 (P<0.001, P<0.001), but not at D0. The expression level of VEGF-mRNA in observation-window group is significantly lower than that in MSCs group and VEGF group at D3 and D6 and the difference is statistically significant, the expression level of VEGF-mRNA in VEGF group is significantly lower than that in MSCs group at D3 and D6 and the difference is statistically significant. There is an interaction effect between different time points and different groups (F=33.552, P<0.001.The third part1.X-ray scanning:the valley-peak ratio of X-ray is significantly different at different time points, the density of valley and peak is much higher at D2, D3, D4, D6, D10, compared with DO (P=0.001, P<0.001, P<0.001, P=0.013, P=0.010, P=0.036), the density of valley and peak is much lower at D16 compared with D1 (P=0.009), the density of valley and peak is much higher atD6, D10, D16 compared with D2 (P=0.038, P=0.036, P<0.001), the density of valley and peak is much higher at D4, D6, D10, D16 compared with D3 (P=0.038, P=0.004, P=0.007, P<0.001).2. After mark the vascular endothelial cells with fluorescent agent and removing part of the epidermis cells in skin, scan the whole-layer of the skin using confocal laser microscopy to display the small vessels perfectly, then restore a three-dimensional structure of the microvascular anastomose in angiosomes in MIMICS 13.0. Conclusions1. Based on the results of preliminary study of the project, successfully improve and make the skin observation-window group on the back of the rats, which can make it possible to observe the vessels and the on-going of their proliferation real-time under direct vision.2. Discovery from the skin observation-window group:the flap model designed in the experiment can effectively induce the proliferation of choke vessels in the perforasomes in both left and right iliolumbar artery, the skin proliferation shows in a double-peak way. The speed of the proliferation of microvessels advances and the degree of proliferation increases significantly when given the intervention of MSCs, while the density of the microvessels increases, the speed of the proliferation of microvessels advances and the degree of proliferation somewhat increases when given the intervention of VEGF.3.The successful application of laser confocal microscopy in restoring a three-dimensional structure of morphology of vessels in the experiment makes it a better approach to research the vessels in physiological state in vivo, compared with gelatin-lead oxide perfusion... |
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