| Objective: The well-known side effects of phenytoin, gingival overgrowth, which is the manifestation of extracellular matrix proliferation, has been applied in various kinds of wound healing process with clinically documented therapeutic results. Neointima formation after balloon angioplasty will result in target vessel restenosis, which is a common problem encountered in clinical practice. Reduced extracellular matrix component has been observed in the restenosis lesion in previous research. In the present study, we intended to investigate the special pharmacological effect of phenytoin on the healing process after ballon injury in rat carotid artery.Method: Study was performed on rat model of balloon-injury in common carotid artery. All rats were divided into two groups: normal diet group and high fat diet group before operation. Vascular injury was established via left commom carotid by a 2F Forgarty arterial embolectomy balloon catheter according to standard method. The right carotid was treated as sham-operation. Rats survived successful operation were divided into different treatment groups: normal diet, normal diet plus Phe, high fat diet and high fat diet plus Phe. On the 4th day, 7th day and 28th day after operation, rats were anesthetized, and blood was harvested for total cholesterol test, then the left carotids and the corresponding part of the right carotids were seperatedafter perfusion with phosphate buffered solution. The arteries were embedded with paraffin, sectioned into 5 um, stained with hematoxylin and eosin, Victoria blue and picrosirius red to visualize cell nuclei, elastic lamina and collagen, respectively. We also performed immunohistochemisty for detecting a-smooth muscle actin (a-SMA) and proliferating cell nuclear antigen (PCNA). The sections were scanned by digital CCD. The intima area, media area, lumen area, the ratio of intima to media, the circumference of external elastic lamina, the rate of restenosis (neointima index), collagen area and collagen density in intima and cell desity were analyzed by image analysis software. Result:1. High fat diet could raise the concentration of total cholesterol in serum (normal diet 1.76±0.22mmol/L vs high fat diet 2.15±0.44mmol/L, p<0.05), but injury could not result in formation of artherosclerotic plaque in carotid of rats fed with high fat diet for 8 weeks.2. There were no neointimas in the carotids of rats with sham-operation. On the 4th day after operation, there were no obvious neointimas in the injury arteries. On the 7th day after operation, the intimas were thickened and mainly consisted of cells surrounded by extracellular matrix. On the 28th day after operation, the intimas were obviously proliferated, while cell density was decreased and extracellular matrix was relatively increased.3. Analysis of Victoria blue stainingOn the 7th day after operation, phenytoin had no apparent effect on intimas thickening: there was no statistic difference in I/M (normal diet group 0.52±0.11 vs normal diet plus Phe group 0.50±0.12, /?0.05) between groups.On the 28th day after operation, phenytoin could inhibit intima proliferation andlumen narrowing: intima area (153604.35±18463.06um2 vs 203934.11 ±54250.16um2,/?<0.01), I/M (1.70±0.08 vs 2.26±0.46,/?<0.05), the rate of restenosis (59.47±3.19% vs 75.86±13.33%, p<0.0\) in normal diet plus Phe group were all less than those in normal diet group, while lumen area (106064.00+ 23569.16um2vs62897.25±34038.61uni2, /?<0.01) was larger than that in normal diet group;intima area (224239.63±11959.84um2 vs 277997.54±38916.58um2, p<0.01) and the rate of restenosis (65.85±7.59% vs 77.67±10.78%,/K0.05) in high fat diet plus Phe group were also less than those in high fat diet group, while lumen area (121229.03±42934.22um2 vs 80139.54±39887.73um2,/?<0.05) was larger than that in high fat group.High fat diet could aggravate intima overgrowth and result in vascular expansive remodeling: the intima area (277997.54±38916.58um2 vs 203934.1 l±54250.16um2, p<0.0\ ) , I/M (3.07±0.49 vs 2.26±0.46, /K0.01) and the circumference of external elastic lamina ( 2376.36±65.15um vs 2098.29±138.94um, p<0.0\ ) in high fat diet group were all greater than those in normal diet group.4. Analysis of HE stainingOn the 7th day after operation, there was no statistic difference in intima cell density between groups (normal diet group 12064.63±3246.62/mm2 vs normal diet plus Phe group 11247.92±3512.04/mm2, p>0.05). On the 28th day after operation, phenytoin could inhibit the increased cell number in intima: cell desity (7218.02±2008.06/mm2 vs 8484.63±1077.29/mm2, /><0.05)in normal diet plus Phe group was smaller than that in normal diet group.5. Analysis of picrosirius red staining detcected by polarized light Phenytoin had no effects on collagen component deposition in intima after earlyvascular injury: on the 7th day after operation, there was no collagen deposited in intima;on the 28th day after operation, under polarized light, collagen could be visualized, most of which were type III collagen, deposited in intima. But there were no statistic difference in collagen area (normal diet group 1493.20+1566.30 pixels/jWvs normal diet plus Phe group 943.50±659.00 pixels/um2, p>0.05) and desity (normal diet group 0.76 + 0.79% vs normal diet plus Phe group 0.70 + 0.63%, p>0.05) .6. Analysis of immunohistochemistyOn the 28th after operation, phenytoin could inhibit cell proliferation: PCNA positive cell counting (9.89±7.63 per 200 magnification vs 23.03± 13.95 per 200 magnification, /K0.01 ) and a-SMA positive cell counting (30.91±20.05 per 200magnification vs 61.81±16.57 per 200magnification,/?<0.01) in normal diet plus Phe group were all less than those in normal diet group.Conclusion: 28 days after vascular injury, phenytoin can decrease the cell number in neointima and promote the synthesis of extracellular matrix which result in diminished neointima thickening. Hypercholesterinemia can aggravate the intima overgrowth and result in the vascular expansive remodeling after vascular injury. |
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