| Backgrounds and objectives: Subarachnoid hemorrhage (SAH) is one of the majordiseases that threat to life. According to statistics, more than three quarters of subarachnoidhemorrhage is caused by the rupture of intracranial aneurysm (IA). At present, the therapyfor ruptured aneurysm is limited to clipping surgery and endovascular interventionaltreatment,and they have risk and certain complications. Therefore, to investigate thepathological mechanism of formation, development and the breakdown of IA, especially todiscuss whether there is the possibility of repair the aneurysm wall have importantmeaning. Smooth muscle cell layer is the main component of arterial wall. Thepathological changes of the layer are the important basis of the aneurysm formation.Vascular smooth muscle cells (VSMC) is an important part of smooth muscle cell layer.VSMC displays no proliferation capacity in normal physiological differentiation state, butwhen the environment changes, VSMC can generate reversible phenotype conversion. Itwill lost the physiological function of normal mature cell and win proliferation capacity.Visiblely, the plasticity of vascular smooth muscle cell wall will is be the basis of theaneurysm repair. The aneurysm formation is a reversible process of damage and repair inan artery, the proliferation and migration of smooth muscle cell in membrane layer areimportant to repair. The studies of pathology have indicated that elastase-inducedaneurysm model is almost consistent with human’s cerebral aneurysm and more closed tothe ideal aneurysm model than models created by other methods. We propose to discussthe possible pathogenesis through investigating proliferation activity of smooth muscle cellin aneurysm wall and provide basis for the repair research of intracranial aneurysm wall. Methods:(1)30New Zealand rabbits(2.5~3.0kg),half male and half female,weredivided into5groups randomly,group A(control group),group B(one week),group C(twoweeks),group D(three weeks), group E(four weeks),feeding each rabbit freely in roomtemperature. No feeding and water for12hours before operation.(2) Measure the diameterof the vessel is which about0.5mm~1.0mm far from the bifurcation of right commoncarotid artery (CCA), dissect the superficial fibrous connective tissue under themicroscope,then drop50u/ml elastase solution0.2ml on surface of the point vessel,dropPBS on control group,inject500u heparin through rabbits posterior auricular vein20minutes later.(3) One week after operation,use CT angiography (CTA) to detect themorphologic change of the CCA and formation of the aneurysms in all rabbits.(4) From theoperation day,execute6rabbits every week. Measure the neck width and altitude of theaneurysms before executing, then collect the aneurysm samples.(5) Using HE dyeing andlight microscope to examine the aneurysm samples.(6) Using immunohistochemistry todetect the expression of the proteinum of α-SMA,PCNA and MMP-2.(7)Another aneurysmsamples were undertaken real-time polymerase chain reaction (PCR) to detect theexpression of the mRNA of OPN and MMP-2.Results:(1) CTA results and measurement: use CTA to detect experimental rabbits on7th day after operation,we found visible fusiform or cystiform aneurysms where theelastase was dropped, but we did not find aneurysms in the control group. Compared withgroup B, the width and altitude of aneurysms of group C is obviously larger(p＜0.05),andthere is no significant difference compared with group C and group D respectively (p＞0.05).(2) Pathological results:the pathological examination of the experimental aneurysmsshowed that the internal elastic layers of aneurysms wall were destructed or disappeared,elastic fibers disrupted, vascular smooth muscle cells became atrophy and less, vascularsmooth muscle cell layer became thinner.(3) Immunohistochemistry results: theimmunohistochemistry results disclose that: compared with tontrol group, the expressionof MMP-2and PCNA was obviously more in experimental groups,while the expression ofα-SMA was obviously less (p＜0.05). The MMP-2expression of C group was thehighest,and it had significant difference compared with other groups (p＜0.05). the PCNAand α-SMA expression gradually decreased in experimental groups (p＜0.05). Comparedwith another three groups respectively,the PCNA expression of group B were significantlyhigher (p＜0.05),but there was no significant difference among another three groups (p＞ 0.05).There was significant difference every two groups about α-SMA expression (p＜0.05).(4) Real-time PCR results:the Real-time PCR detection consequence manifest that:compared with tontrol group, the expression of OPN and MMP-2was obviously more inexperimental groups. the OPN expression gradually increased in experimental groups (p＜0.05). There was significant difference every two groups about α-SMA expression (p＜0.05). The MMP-2expression of C group was the highest,and it had significant differencecompared with other groups (p＜0.05).Conclusions:(1) Dropping elastase on rabbits’ CCA can create visible fusiform orcystiform aneurysms, It is a simple method with low cost and can be repeat easily.Morphological characteristics, histopathology and the mechanism of formation of theaneurysm model were similar to human’s IA. The elastase-induced aneurysm model canbe used to study the etiology and treatment of aneurysm, it is one of the perfect models.(2)In the process of aneurysm formation, vascular smooth muscle cells in aneurysm wall lostthe physiological function of normal mature cell,win proliferation capacity and transformfrom the contractile phenotype to a synthetic phenotype.(3) The decrease of the quantityand layers of vascular smooth muscle cells plays a key role in the process of aneurysmformation. The increase of MMP-2secretion is important cause of the change of vascularsmooth muscle cell layers. Backgrounds and objectives:The rupture of intracranial aneurysm (IA) is theprimary cause of subarachnoid hemorrhage (SAH), its lethiferous and disable ratio are veryhigh.At present,the therapies of IA are limited to clipping sergery and intravascularinterventional treatment, there are very few researches about nonoperative treatments suchas repair for IA. As the development of biological and gene therapy,they will bring wideprospects to repair therapy for IA. Intracranial artery includes endarterium layer, smooth muscle cell layer and adventitia layer. When aneurysm begins to form, endarterium layer isfirst be destroyed, then inner elastic layer fractures and disappears, smooth muscle cellsdegenerate and smooth muscle cell layers reduce, at last adventitia layer is destroyed andIA ruptures. In the whole process of aneurysm formation, the degeneration of smoothmuscle cell layers is particularly significant, and the degeneration is a reversible process ofdestruction and repair. But because of the influence of the blood flow shear stress andinflammation, effect of damage is more than repair, eventually leading to the rupture ofaneurysm. Therefore, discussing proliferation activity of smooth muscle cells in theformation of intracranial aneurysm, will become the basis of repair research for aneurysmwall. Based on the models of rabbits’ common carotid artery aneurysms in the part I,we usethe artificial synthetic E-selectin interference aneurysms in order to observe therelationship between inflammation and proliferation activity of smooth muscle cell inaneurysm wall and provide basis for the repair research of intracranial aneurysm wall.Methods:(1)24New Zealand rabbits, half male and half female,were divided into5groups randomly,group F(control group),group G(one week),group H(two weeks),groupI(three weeks), group E(four weeks),feeding each rabbit freely in room temperature. Nofeeding and water for12hours before operation.(2) Create aneurysm models as part I.(3)Embed24GY intravenous detaining needles in right ear vein for all rabbits of treatmentgroups, then inject2mg/kg artificial synthetic E-selectin through right ear vein in treatmentgroups every week until they were executed.(4)One week after operation, use CTangiography (CTA) to detect the morphologic change of the CCA and formation of theaneurysms in all rabbits.(5) From the operation day, execute6rabbits every week. Measurethe neck width and altitude of the aneurysms before executing, then collect the aneurysmsamples.(6) Using HE dyeing and light microscope to examine the aneurysm samples.(7)Using immunohistochemistry to detect the expression of the proteinum of α-SMA,PCNAand MMP-2.(8)Another aneurysm samples were undertaken real-time polymerase chainreaction (PCR) to detect the expression of the mRNA of OPN and MMP-2.Results:(1)CTA results and measurement: use CTA to detect all the24rabbits0n7thday after operation,we found visible fusiform or cystiform aneurysms where the elastasewas dropped, and we also found that some vessels were obstructed. Compared withexperimental groups, the width and altitude of aneurysms of treatment groupsdecreased.The difference is not significant at1th week(p＞0.05),but it is obvious at another three time points(p＜0.05).(2)Pathological results: Compared with experimental groups,the width and altitude of aneurysms of treatment groups，the reduce of the quantity ofsmooth muscle cells and smooth muscle cells layers was greatly declined, the degree ofbreakage of elastic fiber and degradation of smooth muscle cells was alsodecreased.(3)Immunohistochemistry results: the immunohistochemistry results disclosethat: there were different level reduces of MMP-2expression between experimental groupsand treatment groups at each time point. There was significant difference between group Fand B, group G and C(p＜0.05),while the difference of the other two time points was notsignificant(p＞0.05). The expression of PCNA of group F and G decreased obviouselycompared with group B and C respectively(p＜0.05). But there was significant increase at3th week(p＜0.05).There was no significant difference between group E and I(p＞0.05).There were different level reduces of α-SMA expression between experimental groups andtreatment groups at each time point. The difference was not significant(p＞0.05) at4thweek while The difference was obvious at another three time points(p＜0.05).(4)Real-time PCR results:the Real-time PCR detection consequence manifest that there weredifferent level increases of OPN expression between experimental groups and treatmentgroups at each time point. The difference was not significant at4th week(p＞0.05) whileThe difference was obvious at another three time points(p＜0.05). There were differentlevel reduces of MMP-2expression between experimental groups and treatment groups ateach time point. There was significant difference between group F and B, group G and C(p＜0.05),while the difference of the other two time points was not significant(p＞0.05).Conclusions:(1)The artificial synthetic E-selectin can inhibit the reduction of thequantity of smooth muscle cells and smooth muscle cells layers and alleviate the degree ofbreakage of elastic fiber and degradation of smooth muscle cells, thereby delaying orinhibiting the pathological process and rupture of aneurysms.(2)The artificial syntheticE-selectin can promote the transformation of smooth muscle cells to synthetic phenotype,strengthen the proliferation activity of smooth muscle cells, and then repair the aneurysmwall. |
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