| Part I . Quantitative morphometric analysis of vascular remodeling in rat CCA injury model1. Contraction rate of vessel bar in situ perfusion and fixation was less than 10% which wasclose to the vessel form while alive. However, contraction rate of vessel bar in non- in situ perfusion and fixation was 100%.Compared with each other, the thickness of vessel wall, EELA, the ratio of luminal area (LA) to section of vessel had significant difference.2. Shortly after PTA the thickness of vessel wall was thinned obviously, then turned thick, at 28th day group and the thickness of vessel wall arrived at peak level then decreased. After PTA lh the ratio of luminal areas to section of vessel reached maximum, then the ratio gradually decreased. The ratio of vessel wall areas to section of vessel gradually increased. VRI reached maximum shortly after PTA. It decreased distinct in 3-day group and slight increased in 7-day groups then gradually decreased. The percentage of residual luminal areas changed consistently with VRI.3. After PTA , LA gradually decreased in general, with lh group being increased obviously compared with control group,3-days group being reduced distinctly compared with lh group, and there being no difference between 3-day group and 7-day group, compared with 7-day group, while 14, 28 and 42-day group being reduced distinctly. The neointima areas graduallyselected , with 7 specimens involved in each time point. As control, 7 specimens were selected randomly from non- in situ perfusion group. The 7 specimens were stained by following methods.2. Pathological examination: All specimens were stained with HE, Masson trichrome stain. PCNA α-Smooth-Actin bcl-2 bax were stained with immunohistochemistry. Cell apoptosis was stained with in situ hybridization (ISH) by TUNEL method. Two specimens from each time point oflh, 3, 7,14-day were tested by electronic microscopy to observe the ultrastructure changes.3. Pathological imaging analysis: morphological quantitative analysis was performed on HE, Masson slices. Positive cell number and its percentage were counted in the neointima, media, and adventitia respectively for the PCNA, bcl-2, bax, TUNEL slices.ResultsPart I . Quantitative morphometric analysis of vascular remodeling in rat CCA injury mode!1. Contraction rate of vessel bar in situ perfusion and fixation was less than 10% which wasclose to the vessel form while alive. However, contraction rate of vessel bar in non- in situ perfusion and fixation was 100%.Compared with each other, the thickness of vessel wall, EELA, the ratio of luminal area (LA) to section of vessel had significant difference.2. Shortly after PTA the thickness of vessel wall was thinned obviously, then turned thick, at 28th day group and the thickness of vessel wall arrived at peak level then decreased. After PTA lh the ratio of luminal areas to section of vessel reached maximum, then the ratio gradually decreased. The ratio of vessel wall areas to section of vessel gradually increased. VRI reached maximum shortly after PTA. It decreased distinct in 3-day group and slight increased in 7-day groups then gradually decreased. The percentage of residual luminal areas changed consistently with VRI.3. After PTA , LA gradually decreased in general, with lh group being increased obviously compared with control group,3-days group being reduced distinctly compared with lh group, and there being no difference between 3-day group and 7-day group, compared with 7-day group, while 14, 28 and 42-day group being reduced distinctly. The neointima areas graduallyincreased during 3-14 day groups, but there being no difference among 14-42 day groups. Compared with control group, IELA of lh group increased distinctly, 3-day group reduced distinctly compared with control group and 7-day group, 14t, 28 and 42-day group reduced distinctly. EELA gradually decreased.4. There were negative correlation between VRI and residual restenosis, whereas positive correlation between VRI and LA change. There were no correlation among neointima and residual restenosis and LA.There were positive correlation between adventitia areas / (intima +media) areas and VRI. Changes of EELA and IELA were positive correlated with LA. Part II. Changes of cell proliferation, apoptosis of vessel wall and their relation to VRI 1 .Electronic microscopy examination :lh after PTA exfoliated endothelium cell and apoptosis endothelium cells, VSMC were found, but no proliferation cells, VSMC and fibroblast being showed contractile phenotype.3 days after ,the amount of cells in adventitia increased. VSMC and fibroblast were showed synthesize phenotype, apoptosis VSMC still can be found. During 7-14 day the extra cellular matrix (ECM) was increased, some synthesize phenotype VSMC being changed into contractile phenotype, apoptosis VSMC still can be found in media. Neointima cell was showed synthesize phenotype.2. Apoptosis intima cell was found near injury part. Apoptosis media cell was found under IELA. Proliferation cell in adventitia was mainly found near EELA and outer part of adventitia .Among all layer of vessel wall apoptosis preceded cell proliferation after angioplasty, apoptosis and cell proliferation expression peaked at 3-14 day groups. Double stain of PCNA and a- Actin was performed on proliferation cells which positive dye being found in neointima and media which suggest proliferation VSMC. Noct- Actin positive dye was found in adventitia while PCNA positive dye which suggest they be not proliferation VSMC.3. PCNALI of intima was higher than media. ALI was lower than media. PCNAL1 was higher than ALI in intima, peak of PCNALI being appeared in 7-day group. It still kept high level in 14, 28-day groups and obviously decreased in 42-day group.The peak of ALI appeared in 7-day group and it gradually decreased. The cell amount gradually increased and reached peak at 28-day group. The neointima area gradually increased which was positive correlated with cell amount among it. In media, PCNALI was slighter lower than ALI which changedconsistently. Cell proliferation and apoptosis expression kept high level during 3-14 day groups, reached peak at 7-day group then decreased obviously. The cell amount and area of media kept little change, they were positive correlated with each other.4. PCNALl of adventitia was higher than ALI obviously, ALI kept in low level. The peak of PCNALl appeared in 3 day group, it still kept high level in 7, 14-day group being reduced obviously in 28,42-day groups.The cell amount reached most, then gradually reached normal level. The areas of adventitia reached peak in 7-day group then decreased. There were no beeline correlation between areas and cell amount of adventitia which suggest the main component of adventitia -collagen maybe responsible for the change of adventitia areas.5. In 3-14 day groups, there was positive correlation between ALI and VRI both in intima and media .However, there was negative correlation between ALI and residual restenosis both in intima and media. There was no correlation among PCNALl in all lay of vessel wall and ALI in adventitia to residual restenosis, VRI.Part III. The expression and significance of bcl-2, bax during restenosis1. We can not find bcl-2 expression in control group and lh group in the intima, from 3 day, bcl-2 begin to expressed and gradually increased. In the media, bcl-2 can be found in control group. Compared with control group, no change was found in lh group. In 3-day group it decreased, from 7to42 -day group bcl-2 expression gradually increased.2. We can not find bax expression in control group and lh group in the intima, from 3 day, bax begin to expressed and gradually increased to peak in 7-14 -day groups. In the media, bax can be found in control group. From lh bax begin to expressed and gradually increased to peak in 7-day group then decreased.3. In the intima and media bax/bcl-2 was showed increased before decreased and reached peak in 7-day group.4. There was negative correlation between ALI and bcl-2 exprssion both in intima and media.There was positive correlation between ALI and bcl-2 exprssion, bax/bcl-2 respective both in intima and media.Conclusion:1. To evaluate VR, we create the common carotid artery injury model of rat successful. It canevaluate VR impersonality and accurate when draw the materials from arteries after in situ perfusion and fixation.
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