An Immunohistochemical Study On The Expressions Of β-amyloid,α-actin,collagen Ⅳ In Cerebral Vessels With Unknown Type Of Pathological Lesion

IntroductionBlood flowing into subarachnoid space after angiorrhexis is called subarach-noid hemorrhage (SAH). SAH is frequently encountered in the medico -legal investingation and accounted for 90% of intracranial hemorrhage. SAH consists of two types: traumatic SAH and untraumatic SAH, which mainly result from break of weak vessels and consequence intracranial bleeding. Recently, it has been found that some unknown structural abnomalities of small vessels in the superficial layer of cortical or subarachnoid space accompanied with SAH characterized by substitution of homogeneous and pinkish substances for smooth muscle cells when stained with hematoxylin and eosin in forensic autopsy cases. Pathological changes of small vessels are different from those of vascular sclerosis and Cerebral amyloid angiopathy (CAA). There are no such kinds of relevant articles recorded in the literature. It is yet unknown about its pathogenesis and his-topathological categorization.CAA is considered the third most common cause of spontaneous intracere-bral hemorrhage resulted from hypertension and subarachnoid aneurysmal hemorrhage. Smooth muscle cells of CAA vessels are substituted for homogeneous and pinkish substances,which mainly consist of 4kD β -amyloid ( Aβ) . Aβ generation depends on proteolytic cleavage of the amyloid precursor protein ( APP). Deposition of Aβ mainly locates in the wall of small to medium - sized blood vessels in the cortical and leptomeninges. The clinical characters of CAA are multi - lobes bleeding and rebleeding. Aβ can decrease vessel smooth muscel cell ( VSMC) viability by disrupting VSMC -extracellular matrix (ECM) adhesion. Aβ may activate apoptotic pathways to cause loss of VSMC in CAA. Substitutionof vessel smooth muscles for amyloid weakens the structure of vessels which results in bleeding.Vessel smooth muscle cells ( VSMCs) are located in tunica media, and are the important factors responsible for vessel activities, vessel construction and maintainance of angiotasis. According to structures and functions, VSMC may be divided into two phenotypes: contraction type which can differentiate but not proliferate and synthesis type which can not differentiate, actin is the most abundant intracellular protein in the eukaryoticcell and consists of three subtypes;a - actin ^p - actin ^-y -actin, among which a -actin is mainly located in muscle cells, a - actin in vessel smooth muscle cells is called smooth muscle - a - actin ( SM - a - actin). SM - a - actin expression in SMCs is complex and maybe involved in the formation of a unique multiprotein initiation complex that is coordinated by serum reacting factors (SRF) complexes bound to multiple CArG elements. Expression of SM - a - actin is a mark of activating VSMC. Enhanced expression of SM - a - actin is a sign of transformation from synthesis type to contraction type. The expression of SM - a - actin varies with different kinds of cerebrovascular diseases.The extracellular matrix surrounds cells and contributes to the control of cellular function and is involved in the maintenance of the cell' s differentiated state, collagen IV is located in the vascular basement membrane and surrounds each smooth muscle cell. Type IV collagen may be classified as a protein family of triple helical isoforms which can promote sticking, migration, differentiation and growth of cells. Type IV collagen consists of six genetically distinct chains;the classical od(lV) and a2( IV) chains and a3( IV) , a4( IV) , a5( IV) , and ct6( IV) chains. Isoforms producted in vascular wall are mainly cd( IV) and a2 (IV) chains. The expressions of collagen IV are different in vascular sclerosis, artherosclerosis, CAA and aged individual' s cerebral vessels.We have studied the expressions of (3 - amyloid, a - actin and collagen IV in cerebral small vessels with an unknown type of pathological changes through pathohistological staining techniques and immunohistochemical staining techniques, to comprehend the structural characteristics of the vessels , ascertain their histopathological categorization and give convenience to further studies.Materials and methods1. Main ReagentsMouse anti human a - actin ( smooth muscle) monoclonal antibody ( MAB - 0003 ) , Mouse anti human collagen IV monoclonal antibody ( MAB - 0025) , Mouse anti human f$ - amyloid monoclonal antibody ( MAB - 0505 ) and SP im-munhistochemisry Kit ( Kit - 9701) were purchased from Fuzhou Maxim - Biological Technology Ltd. DAB chromogen ( ZLI - 9032 ) were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd.2. Materials20 paraffin embedded brain specimens were obtained from forensic medicin college of China Medical University. Vasculopathy group: 10 specimens were obtained from 10 patients suffered from defferent grades of SAH (the age ranges from 35 -70 years old ( average 45.7 ± 10) ) , whose smooth muscle cells of tunica media in the cortical or subarachnoid space were disappeared, and substituted by homogeneous and pinkish substances when stained with hematoxylin and eosin. Control group: paraffin embedded brain specimens were obtained from 10 patients with normal cerebral vasculars (the age ranges from 25 - 45 years old ( average 40 ± 6 ) ).Methods3.1 Specimen treatmentBrain tissues were cut form frontal lobe and temporal lobe, which were main bleeding areas in brain, and fixed in 4% buffer formalin and then embedded in paraffin. 5 - jxm - thick serial sections were cut from each paraffin block of each piece of brain tissue.3.2 The HE staining were conducted as normal procedures3.3 The congo red staining were conducted as follows: [ 1 ] The sections were deparaffinized in xylene;[2] Hydrated in the gradient alcohol form 100% to 70% , each 5 min;[ 3 ] Washed in redistilled water for 5 min;[4] Stained with hematoxylin for 10 min;[5] Differentiated with 1% hydrochloric acid alcohol for 5 -6s;[6] Rinsed in distilled water for 10 min;[ 7 ] Stained with 1 % congo red for 20 min;[8] Differentiated with 100% alcohol;[9] Deparaffinized in xylene, sealed with neutral gum. 3.4 The immuneostaining were conducted as follows:[ 1 ] The sections were deparaffinized in xylene.[2] Hydrated in the gradient alcohol form 100% to 70% , each 5 min;[ 3 ] Washed in redistilled water for 5 min, and in PBS 5 min;[4] Antigen unmasking: sections for immunostaining with mouse anti human a -actin( smooth muscle) monoclonal antibody do not require special pre-treatment. Sections for immunostaining with mouse anti human collagen IV monoclonal antibody were digested in 0.4% pepsin at 37°C for 30 min. Sections for immunostaining with mouse anti human p - amyloid monoclonal antibody were pretreated with high -pressure technique in 0. 01M , PH6. 0 citrate buffer;[5] Put sections in the 3% H2O2 PBS (135ml PBS +30% H2O2 15ml) at room temperature for 20 min, for inhibition of endogenous peroxidase;[ 6 ] Washed in PBS 5 min 3;[ 7 ] Incubated with non - immune goat serum at room temperature for 20 min;[ 8 ] Incubated with mouse anti human a - actin ( smooth muscle) monoclonal antibody or mouse anti human collagen IV monoclonal antibody or mouse anti human (3 - amyloid monoclonal antibody at 1:100 titer at room temperature for 60 min;[9] Washed in PBS 5 min x3;[10] Reacted with second antibody, at room temperature for 20min;[11] Washed in PBS 5 min 3;[12] Incubated with the SP reagent at room temperature for 20min;[ 13 ] Washed in PBS 5 min 3;[14] Visualized with DAB at room temperature for 3-10 min.;[ 15 ] Washed in tap water for 5 min and then in distilled water for 2 min;[16] Stained with hematoxylin for 10 min.[17] Differentiate with 1% hydrochloric acid alcohol for 5 -6s.[18] Differentiate in tap water for 10 min;[19] Dehydrated, and mounted with neutral balsam.4. Evalution of the resultsThe amyloid appears pale salmon - pink when stained with congo red and shows characteristic apple - green birefringence under polarized light microscope.P - amyloid ^ a - actin and collagen IV were detected immunohistochemical-ly in cells with brown - stained plasma. Qualitative analysis;The results were classified according to the intensity of the staining: - as Negative, + as positive, + + as moderately positive, + + + as intensive positive. Semiquantita-tive analysis: We used image analysis system ( Motic) (200 fold) to measure average grey density of the vascular walls and the background and calculate the ratio of average grey density of vascular walls to the grey density of background in percentage.Statistical analysis -.The statistical analysis was conducted by SPSS 10. 0 for Windows and sum of ranks was used for comparison, with P < 0.05 regarded as the significant difference.Results1. HE stainingAbnomal small vessels were located in subarachnoid space and pallium. The walls of vessels were thickened, but the lumens were not narrowed. Structures of tunica media and adventitia were abmomal, endo - elastic layer and smooth muscles almost totally disappeared and substituted by homogeneous and pinkish substances, more seriously, only endothelium remained.2. Congo red stainingThere were no apple - green birefringence under polarized light microscope in both groups, while vessels in leptomeninges of CAA samples appeared charac-teristic apple - green birefringence .3. Immunohistochemical staining3.13- amyloid immunohistochemical stainingThere were no positive expressions of p - amyloid in both groups, while vessels in leptomeninges of CAA samples appeared positive expressions. There was no difference between two groups (p >0.05).3.2a- actin immunohistochemical stainingParts of abnormal vessel tunica media expresseded a - actin negatively, parts expressed a positive ring form under the endothelium, and parts appeared a few and interrupted positive expression in tunica media. Tunica media of control group showed positive expression. There was statistical difference between two groups (p <0. 05) , the expression of a - actin in abnormal vessels was fewer than in normal vessels.3.3 collagen IV immunohistochemcal stainingAbnormal vessels expressed a positive ring form under the endothelium. Some showed interrupted ring form. Some individuals showed negative expression. Parts of abnormal vessel tunica media expressed collagen IV negatively, parts showed a few interrupted positive expressions. Endothelium and tunica media of normal vessels showed positive collagen IV expressions. There were statistical difference between two groups (p <0.05) , the expression of collagen IV in abnormal vessels was fewer than in normal vessels.DiscussionThere were some similarities between CAA and the abnormal cerebral small vessels: they both located in subarachnoid space or cortex;VSMCs were disappeared and substituted by homogeneous and pink substances when stained with hematoxylin and eosin;the expression of a - actin in vessel tunica media was negative or partly positive, which suggested the damage of VSMC. While there were some obvious differences between them: (1) individuals suffering from the unknown type of the pathological change of the cerebral small vessels were younger than those of CAA;(2) vessel tunica media expressed collagen IV nearlynegatively or partly positively, which suggested that there be little collagen IV in homogeneous and pink substances;( 3 ) the abnormal vessels showed no apple -green birefringence under polarized light microscope when stained with congo red and expressed Ap negatively with immunohistochemical staining, which suggested that the vessel walls contain no A(3.We found no Arteriolosclerosis in any organ in the patients suffering from unknown type of pathological lesion of cerebral small vessels. In these cases, abnormal cerebral small vessels were mainly located in subarachnoid space and cortex, which was different from the distribution of Arteriolosclerosis. The vascular walls were thickened but the lumens were not narrowed, and more seriously , only endomembrane were remained, which was different from Arteriolosclerosis in morphology. The expression of a - actin in abnormal vessels was similiar with Arteriolosclerosis, but the collagen IV expression was decreased and most of it located in basal membrane, which was different from that of Arteriolosclerosis.VSMCs were disappeared in cerebral small vessels with the unknown type of pathological lesion. As for the vessels with mild pathological changes, the expressions of SM - a - actin and collagen IV were positive in a granular - and -ring form under the basal membrane, and positive in interrupted line form or negative in vessel tunica media;in severe case, the positive expression can only be seen in the basal membrane. All these findings mentioned above suggested that the damage of VSMCNSM - a - actin and collagen IV be parellel processed and subsequently exhibits the damage of SM - a - actin and collagen IV in basal membrane indicating that the pathological changes may reveal a trend of centripetal development.It was because of the changes of activities or the relative secretory volume of MMPs and TIMP that made the decomposition of collagen IV increased , which made collagen IV decreased or even disappeared. Many factors operating together or singly resulted in VSMC proliferation blocking, Apoptosis or death in advance, which made VSMC decreased or disappeared.We found in forensic autopsy cases that VSMC almost disappeared and collagen IV obviously decreased in some cerebral small vessels with the unknownpathological change which resulted in the vascular wall weakened and disrupted and bled when the press fluctuation or other non - external force took place. As the abnormal vessels located in subarachnoid space or cortex adjacent, blood was easy to flow into the subarachnoid space when vessels were broken, and thus led to SAH.ConclusionBy pathohistological and immunohistochemical staining techniques, we have studied an unknown type of pathological lesion in cerebral small vessels in the dead persons suffering from untraumatic SAH. The results were as follows;1. Among the cases of the unknown type of pathological lesion, the patients ' ages and vascular wall' s components were obviously different from those of CAA. Cerebral small vessels of the case had no p - amyloid, and only a few of collagen IV was in tunica media.2. The locations, morphological characteristics and vascular wall' s components of the abnormal vessels differed from those of hyaline arteriolosclerosis. The abnormal vessels were located in subarachnoid space and cortex;vessel walls were thickened, and the lumens were not narrowed;collagen IV in tunica media were obviously decreased.3. VSMCs in tunica media of cerebral small vessels with the unknown type of pathological change were almost disappeared;the expressions of a - actin and collagen IV were obviously decreased, especially in tunica media;lesions of VSMC, a - actin and collagen IV were synchronous, which revealed a trend of centripetal development.