Comparative Study Between High Resolution MR Imaging With Pathology And Molecular Pathological Study Of The Carotid Atherosclerotic Plaques

BACKGROUND:The risk of CAS to cerebral ischemic events is not only dependent on the stenosis of the artery lumen, but more dependent on the stability of the plaque structure and the heterogeneity of the intraplaque components. With the development of the imaging technique and instrument, the high resolution MR imaging quality and its display effect to the CAS plaques have approved rapidly, but the comparative study between the imaging and the pathology of CAS plaques goes on slowly. In addition, the pathological study demonstrats that CAS is a series of chromatic inflammatory process of the artery wall, many inflammatory factors involved in the processes of inflammatory infiltration, tissue necrosis, microvessel formation, fibrosis and calcification during the CAS plaque progression, and play roles in the plaques destability. Recent research have found that HMGB-1, a kind of non-histone DNA binding protein in the nunclear, expressed more and released to the extracellular matrix under multiple pathological condition, and being a late inflammatory factor, play a role in the regulation of EC and SMC function and the other inflammatory factors' expression during the AS progression. But there is no systemic in vivo research on the expression and distribution of HMGB-1 in human CAS plaques. PURPOSE:This research includes two separate parts. Part I aims to investigate the high resolution MRI signal characteristics of the fibrous cap and the intraplaque components include lipid pool, necrotic core, calcification, hemorrhage, and fibrosis through the comparative analysis of the multiple sequence MR imagings with the pathological characteristics. In oder to provide basis for clinical especially the pre-operation determination of plaque stability. Part II aims to investigate the expression and distribution of HMGB-1 in CAS plaques through the detection of the protein and mRNA of HMGB-1. METHODS:The plaques come from 32 CAS patients (mean age 63.8 years;28 males) scheduled for CEA operation during January 2004 to January 2006, who wereimaged with a 1.5-T scanner using A standardized protocol to obtain 4 different contrast weighted images (T1WI, PDWI, T2WI and 3D TOF) of the carotid arteries. Carotid plaques were removed and processed for histological examination. In Part I, 25 cases are choosed;the plaques are taken photoes under microscope, and then fixed up to make paraffin slices and processed H-E, Masson, Mallory and tri-collagen staining. Based on the hisological characteristics, plaques are classified and the MRI imaging characteristics of the fibrous cap and the intraplaque components include lipid pool, necrotic core, calcification, hemorrhage and fibrosis are analyzed. In part II, all paraffin slices are processed immunohistochemical staining of HMGB-1, and the other 7 cases are processed to gather protein and mRNA respectively for western blot and RT-PCR in order to analyze the expression and distribution of HMGB-1. RESULTS:In the research, 32 plaques are gathered through CEA operation, more from men (28 cases) than women (4 cases), there are no sexual differences between age, lateral and length and the plaques are mainly situated in the CCA bifurcations.In Part I25 plaques are sliced up and 248 slices (72.37%) have their corresponding MRI imaging and used for comparative study. All slices are classified according to AHA pathological classification and MRI classification, there is a high coherence between the two classification with a Cohen's k (95% confidence interval) of 0.782 (0.694 to 0.869).The major histological changes of fibrous cap are intima thickness and laminar sub-intima fibrosis;MR imaging showing a coiled or crescent hypo-intense signal surrounding the lumen on 3D TOF, the thickness and consistency of signal indicate the thickness and intactness of fibrous caps.Lipid pool pathologically manifested a mass or zonal conglomeration of lipid vacuole, while necrotic core manifested a mixed of tissue necrosis, nuclear conglomeration, fiber debris, shapelessly matrix deposit, inflammatory infiltration, and varied edema in the surrounding hyperplastic fibrosis. It is difficult to differentiate them on MRI for them all show hyper-intense signals on T1WI, PDWI, and and T2WI images with slightly hyper-intense signal on 3D TOF images.Calcification mainly manifested sheets of tissue loss and radially arrangedcrystalline-like substance pathologically, with a border of necrosis, inflammatory infiltration and/or fibrosis, on MR imaging it usually shows fair bordered arc or irregular hypo-intense signals on all 4 sequences, some diffused calcification may be intermingled with necrotic and fibrosis and manifested mixed signals.The pathological and MRI expression of Intraplaque hemorrhages differ with time and stage. Fresh hemorrhages mainly contain intact red cell and presents jacinth on Masson and Mallory staining, the MR images show slight hyper-intense to hyper-intense signals on T1WI and 3D TOF imaging, and iso-intense to slight hyper-intense signals on T2WI imaging, while varied signals on PDWI imaging. Recent hemorrhages mainly contain fragmentary red cells with congregation of fibrin, crystal cholesterol, and macrophages, and presents brick-red to brown on Masson and Mallory staining, the MR images show hyper-intense to slight hyper-intense signals on all 4 sequences. Old hemorrhages mainly consist of a collection of shapeless necrotic debris with mild inflammatory infiltration, and show green and blue respectively on Masson and Mallory staining, the MR images show hypo-intense signals on all 4 sequences.Fibrosis characterize a college hyperplasia with varied neoangiogenesis and smooth muscle cell hyperplasia in deep plaques and the superficial shoulders pathologically, the MR images mainly show varied signals on all 4 sequences, predominant by iso-intense signals, and some may shown hyper-intense signals on PDWI and T2WI.In Part IIImmunohistochemistry staining show that there is HMGB-1 expression on normal artery walls symmetrically distributed and mainly situated in the nuclears of ECs, SMCs and fibroblast cells. In the plaques, there is a hyper expression of HMGB-1, mainly situated in the nuclears and cytoplasms of monocytes and macrophages in inflammatory areas. In the intercellular matrix on the border of necrotic and calcification areas, there is a HMGB-1 deposit. There is a certain liner correlation between the ratios of HMGB-1 positive areas to inflammatory areas and the counts of monocytes and macrophages.Western Blot and RT-PCR coincidently show that HMGB-1 protein and mRNA express the highest in necrotic areas, higher in calcification areas, and low in fibrosis and Lipid pool areas. CONCLUSIONS:High resolution MRI multiple sequences contrast analysis can not only show the CAS plaque configuration but also differentiate the intraplaque components. 3D TOF imaging shows the fibrous cap better with the potency to distinguish the thickness and integrity. Multiple sequences contrast analysis can clearly identify the intraplaque calcification and hemorrhage and discriminate the hemorrhage time and stage. The discrimination of lipid pool and necrotic core on MRI is difficult for both mainly showing hyper-intense signals on all 4 sequences. Fibrosis usually shows iso-intense signals on all 4 sequences, sometimes hyper-intense signals on PDWI and T2WI imaging.The expression of HMGB-1 increases in CAS plaques and is mainly distributed in the areas of inflammatory infiltration and the border of necrotic and calcification areas, and mainly situated in the nuclears and cytoplasms of monocytes, macrophages and ECs. There is a certain liner correlation between The HMGB-1 hyper-expression and the inflammatory infiltration. All these indicate that HMGB-1, being a late inflammatory factor, may play a role in the pathological progress and development of CAS.