Cerebral Aneurysms In Rats Research Articles

Suppression of cerebral aneurysm formation in rats by a tumor necrosis factor–α inhibitor

Although cerebral aneurysmal subarachnoid hemorrhage is a devastating disease for humans, effective medical treatments have not yet been established. Recent reports have shown that regression of some inflammatory-related mediators has protective effects in experimental cerebral aneurysm models. This study corroborated the effectiveness of tumor necrosis factor-α (TNF-α) inhibitor for experimentally induced cerebral aneurysms in rats. Five-week-old male rats were prepared for induction of cerebral aneurysms and divided into 3 groups, 2 groups administered different concentrations of a TNF-α inhibitor (etanercept), and 1 control group. One month after aneurysm induction, 7-T MRI was performed. The TNF-α inhibitor groups received subcutaneous injection of 25 μg or 2.5 μg of etanercept, and the control group received subcutaneous injection of normal saline every week. The TNF-α inhibitor administrations were started at 1 month after aneurysm induction to evaluate its suppressive effects on preexisting cerebral aneurysms. Arterial circles of Willis were obtained and evaluated 3 months after aneurysm induction. Rats administered a TNF-α inhibitor experienced significant increases in media thickness and reductions in aneurysmal size compared with the control group. Immunohistochemical staining showed that treatment with a TNF-α inhibitor suppressed matrix metalloproteinase (MMP)-9 and inducible nitric oxide synthase (iNOS) expression through the luminal surface of the endothelial cell layer, the media and the adventitia at the site of aneurysmal formation, and the anterior cerebral artery-olfactory artery bifurcation. Quantitative polymerase chain reaction also showed suppression of MMP-9 and iNOS by TNF-α inhibitor administration. Therapeutic administration of a TNF-α inhibitor significantly reduced the formation of aneurysms in rats. These data also suggest that TNF-α suppression reduced some inflammatory-related mediators that are in the downstream pathway of nuclear factor-κB.

Dysregulated Expression Profiles of MicroRNAs of Experimentally Induced Cerebral Aneurysms in Rats

ObjectiveCerebral aneurysm (CA) is an important acquired cerebrovascular disease that can cause catastrophic results. MicroRNAs (miRNAs) are small non-coding RNAs, playing essential roles in modulating basic physiologic and pathological processes. Currently, evidences have been established about biologic relationship between miRNAs and abdominal aortic aneurysms. However, biologic roles of miRNAs in CA formation have not been explained yet. We employed microarray analysis to detect and compare miRNA expression profiles in late stage of CA in rat model.MethodsTwenty-six, 7-week-old male Sprague-Dawley rats underwent a CA induction procedure. The control animals (n=11) were fed a normal diet, and the experimental animals (n=26) were fed a normal diet with 1% normal saline for 3 months. Then, the rats were sacrificed, their cerebral arteries were dissected, and the five regions of aneurysmal dilation on the left posterior communicating artery were cut for miRNA microarrays analysis. Six miRNAs (miRNA-1, miRNA-223, miRNA-24-1-5p, miRNA-551b, miRNA-433, and miRNA-489) were randomly chosen for validation using real-time quantitative PCR.ResultsAmong a set of differentially expressed miRNAs, 14 miRNAs were over-expressed more than 200% and 6 miRNAs were down-expressed lower than 50% in the CA tissues.ConclusionThe results show that miRNAs might take part in CA formation probably by affecting multiple target genes and signaling pathways. Further investigations to identify the exact roles of these miRNAs in CA formation are required.

Ibudilast Inhibits Cerebral Aneurysms by Down-Regulating Inflammation-Related Molecules in the Vascular Wall of Rats

Phosphodiesterase-4 (PDE-4) is a cyclic adenosine monophosphate-specific enzyme involved in various inflammatory diseases. We studied its role in and the effect of ibudilast, which predominantly blocks PDE-4, on rat cerebral aneurysms. Cerebral aneurysms were induced at the anterior cerebral artery-olfactory artery bifurcation of female rats subjected to hypertension, increased hemodynamic stress, and estrogen deficiency. The effect of ibudilast (30 or 60 mg/kg/d for 3 months) on their cerebral aneurysms was studied by morphological and immunohistochemical assessment and quantitative real-time polymerase chain reaction assay. In our in vitro study, we grew endothelial cells stimulated by angiotensin II under estrogen-free conditions and examined the effect of ibudilast on PDE-4 activation and the cyclic adenosine monophosphate level. Morphological evaluation using vascular corrosion casts showed ibudilast significantly suppressed cerebral aneurysms in a dose-dependent manner. In rats with induced cerebral aneurysms, the gene and protein expression of PDE-4 was high, and endothelial leukocyte adhesion molecules (P-selectin, intracellular adhesion molecule 1, and vascular adhesion molecule 1), matrix metalloproteinase-9, and tumor necrosis alpha were expressed. Macrophage migration was also increased. Treatment with ibudilast down-regulated these molecules, suppressed macrophage migration into the aneurysm wall, and inhibited PDE-4 activation and the elevation of cyclic adenosine monophosphate in endothelial cells. These results suggest that blocking of PDE4 is associated with the reduction of inflammation-related molecules and macrophage migration, thereby reducing the progression of cerebral aneurysms. It may represent a new conservative therapy to treat patients with cerebral aneurysms.

Effect of olmesartan and pravastatin on experimental cerebral aneurysms in rats

The major initiation process of intracranial aneurysms is thought to involve endothelial dysfunction due to hemodynamic stress. Angiotensin II type 1 receptor blockers and statins improve vascular endothelium function. The effects of olmesartan and pravastatin were investigated on the development of experimental aneurysms in rats. Eighty-three rats underwent aneurysm induction. Seven groups of 10–14 rats were treated with low or high dose olmesartan, low or high dose pravastatin, low doses of olmesartan and pravastatin, hydralazine, or no drug (control) for 12 weeks, when rats were sacrificed for vascular corrosion casting and scanning electron microscopy. Aneurysmal changes at the anterior cerebral–olfactory artery bifurcation were divided into stages 0 (no abnormality) to III (saccular aneurysm). Systolic arterial blood pressure was elevated over 170 mm Hg in the control, low dose pravastatin, and high dose pravastatin groups, but not in the other groups. The control group demonstrated aneurysmal changes in 100% and stage III in 50% of rats. Aneurysmal changes were observed in most rats in the other groups, but the incidence of stage III was 10% or less. The staging pattern showed significant differences between the groups ( P = 0.028). Pravastatin reduced both stages III and II + III and olmesartan ameliorated stage III, implying that these may prevent aneurysmal formation through acting on different steps. (209 words)

0220 血流の物理的刺激がラット誘発脳動脈瘤初期段階の血管壁に及ぼす影響(GS3:循環系のバイオエンジニアリング)

0220 血流の物理的刺激がラット誘発脳動脈瘤初期段階の血管壁に及ぼす影響(GS3:循環系のバイオエンジニアリング)

Analyses of MMP-2,-9 for early stage of induced cerebral aneurysms in rats

嚢状脳動脈瘤の発生についてはマトリックスメタロプロテアーゼ(MMP)ファミリーであるMMP-2,-9の関わりが知られているが，発生初期におけるMMP-2,-9の詳細な解析の報告はない．したがって，脳動脈瘤に対する治療を開発する上で発生機構の解明は必要不可欠である．そこで，片側総頸動脈結紮による脳動脈瘤誘発ラットを使用し，脳動脈瘤発生初期におけるMMP-2,-9の挙動を脳血管組織の免疫染色およびRT-PCRにより解析した．MMP-2は誘発術施行の有無にかかわらず遺伝子発現が認められ，術後の経過でも発現量の変動も認められなかった．MMP-9は術後4週間より非結紮側血管分岐部に遺伝子発現を確認した．これらの結果より，MMP-2遺伝子は発生段階での関与は定かではないが定常的な発現がみられ，脳動脈瘤発生過程でのMMP-9遺伝子の発現が加わることで両者が協調して動脈瘤成熟に関与すると推察される．

Response to Letter by Paraskevas

HomeStrokeVol. 39, No. 8Response to Letter by Paraskevas Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBResponse to Letter by Paraskevas Hiroharu Kataoka, MD, PhD Hiroharu KataokaHiroharu Kataoka Department of Neurosurgery, Kyoto University Graduate School of Medicine, Kyoto, Japan Search for more papers by this author Originally published3 Jul 2008https://doi.org/10.1161/STROKEAHA.107.522011Stroke. 2008;39:e135Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: July 3, 2008: Previous Version 1 Response:We have recently demonstrated that simvastatin suppresses the progression of experimentally induced rat cerebral aneurysms (CA). Stains are used in the prevention and treatment of various cardiovascular and cerebrovascular diseases. Large clinical trials demonstrated the efficacy of statins in preventing cardiovascular and cerebrovascular events,1–4 whereas there are no randomized controlled trials proving statins’ beneficial effects on aortic abdominal aneurysm and CA so far.A clinical trial investigating the preventive effect on CA rupture is difficult because of a low incidence of CA rupture. Only <1% of unruptured CA lead to subarachnoid hemorrhage in 1 year.5 If CA rupture would be set as a primary end point, a tremendous number of patients would need to be enrolled in a study. Such a clinical trial encompasses an ethical problem. Subarachnoid hemorrhage has a high mortality, and surgical clipping and endovascular coiling can prevent CA rupture. To overcome the difficulties described above, we must identify a predictive factor for the stability of CA walls.Positive results obtained from an animal experiment cannot be directly applied to human disease. However, a series of our studies using an experimentally induced CA model provide evidences that vascular inflammation and resultant endothelial dysfunction contribute to degenerative changes in CA walls.6,7 We hope our results will be a clue to the development of a less invasive treatment for preventing subarachoid hemorrhage.DisclosuresNone.1 Heart Protection Study Collaborative Group. MRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in 20 536 high-risk individuals: a randomised placebo-controlled trial. Lancet. 2002; 360: 7–22.CrossrefMedlineGoogle Scholar2 Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet. 1994; 344: 1383–1389.MedlineGoogle Scholar3 The Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) Study Group. Prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. N Engl J Med. 1998; 339: 1349–1357.CrossrefMedlineGoogle Scholar4 Sacks FM, Pfeffer MA, Moye LA, Rouleau JL, Rutherford JD, Cole TG, Brown L, Warnica JW, Arnold JM, Wun CC, Davis BR, Braunwald E; Cholesterol and Recurrent Events Trial investigators. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. N Engl J Med. 1996; 335: 1001–1009.CrossrefMedlineGoogle Scholar5 International Study of Unruptured Intracranial Aneurysms Investigators. Unruptured intracranial aneurysms–risk of rupture and risks of surgical intervention. N Engl J Med. 1998; 339: 1725–1733.CrossrefMedlineGoogle Scholar6 Aoki T, Kataoka H, Morimoto M, Nozaki K, Hashimoto N. Macrophage-derived matrix metalloproteinase-2 and -9 promote the progression of cerebral aneurysms in rats. Stroke. 2007; 38: 162–169.LinkGoogle Scholar7 Aoki T, Kataoka H, Shimamura M, Nakagami H, Wakayama K, Moriwaki T, Ishibashi R, Nozaki K, Morishita R, Hashimoto N. NF-kappaB is a key mediator of cerebral aneurysm formation. Circulation. 2007; 116: 2830–2840.LinkGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails August 2008Vol 39, Issue 8 Advertisement Article InformationMetrics https://doi.org/10.1161/STROKEAHA.107.522011 Originally publishedJuly 3, 2008 PDF download Advertisement

Statins Suppress the Development of Aneurysms: Fact or Fiction?

HomeStrokeVol. 39, No. 8Statins Suppress the Development of Aneurysms: Fact or Fiction? Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBStatins Suppress the Development of Aneurysms: Fact or Fiction? Kosmas I. Paraskevas, MD, FASA Kosmas I. ParaskevasKosmas I. Paraskevas Department of Vascular Surgery, “Red Cross” Hospital, Athens, Greece, Honorary Clinical Research Fellow, Department of Clinical Biochemistry (Vascular Disease Prevention Clinic), Royal Free Hospital, London, UK Search for more papers by this author Originally published3 Jul 2008https://doi.org/10.1161/STROKEAHA.107.521070Stroke. 2008;39:e134Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: July 3, 2008: Previous Version 1 To the Editor:A recent study on rats demonstrated that statins may suppress the development of cerebral aneurysms.1 Similar results were reported in 2 independent experimental studies investigating the effects of statin therapy on abdominal aortic aneurysms.2,3 Compared with placebo, treatment with simvastatin reduced both the overall extent of aortic dilatation, as well as the incidence of abdominal aortic aneurysm development.2,3So far, the beneficial effects of statins on abdominal aortic aneurysm progression in humans have only been supported in 2 observational studies including 1304 and 1505 patients, respectively; regarding cerebral aneurysms, there is no similar report up-to-date.Verification of the positive results from animal studies in randomized controlled trials on humans holds implications for the medical management of both abdominal aortic6 and cerebral1 aneurysms.DisclosuresNone.1 Aoki T, Kataoka H, Ishibashi R, Nozaki K, Hashimoto N. Simvastatin suppresses the progression of experimentally induced cerebral aneurysms in rats. Stroke. 2008; 39: 1276–1285.LinkGoogle Scholar2 Kalyanasundaram A, Elmore JR, Manazer JR, Golden A, Franklin DP, Galt SW, Zakhary EM, Carey DJ. Simvastatin suppresses experimental aortic aneurysm expansion. J Vasc Surg. 2006; 43: 117–124.CrossrefMedlineGoogle Scholar3 Steinmetz EF, Buckley C, Shames ML, Ennis TL, Vanvickle-Chavez SJ, Mao D, Goeddel LA, Hawkins CJ, Thompson RW. Treatment with simvastatin suppresses the development of abdominal aortic aneurysms in normal and hypercholesterolemic mice. Ann Vasc Surg. 2005; 24: 92–101.Google Scholar4 Sukhija R, Aronow WS, Sandhu R, Kakar P, Babu S. Mortality and size of abdominal aortic aneurysm at long-term follow-up of patients not treated surgically and treated with and without statins. Am J Cardiol. 1996; 97: 279–280.Google Scholar5 Schouten O, van Laanen JH, Boersma E, Vidakovic R, Feringa HH, Dunkelgrun M, Bax JJ, Koning J, van Urk H, Poldermans D. Statins are associated with a reduced infrarenal abdominal aortic aneurysm growth. Eur J Vasc Endovasc Surg. 2006; 32: 21–26.CrossrefMedlineGoogle Scholar6 Paraskevas KI, Liapis CD, Hamilton G, Mikhailidis DP. Are statins an option in the management of abdominal aortic aneurysms? Vasc Endovascular Surg. 2008; 42: 128–134.CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Paraskevas K (2009) Regarding “The benefit of statins in non-cardiac vascular surgery patients”, Journal of Vascular Surgery, 10.1016/j.jvs.2009.02.213, 49:6, (1636), Online publication date: 1-Jun-2009. August 2008Vol 39, Issue 8 Advertisement Article InformationMetrics https://doi.org/10.1161/STROKEAHA.107.521070PMID: 18599806 Originally publishedJuly 3, 2008 PDF download Advertisement

Gene Expression during the Development of Experimentally Induced Cerebral Aneurysms

Cerebral aneurysms are a major cause of subarachnoid hemorrhage, but the mechanism of their development remains unclear. In this study, we investigated candidate genes whose expression is significantly changed during the development of experimentally induced cerebral aneurysms using adaptor-tagged competitive polymerase chain reaction (ATAC-PCR). Twenty-four rats received sham operation (control) or the operation for the induction of experimental cerebral aneurysms. Rats were sacrificed at time 0, 2 weeks, 1 month and 3 months after the operation (n = 6 for each group). RNAs from right anterior cerebral artery/olfactory artery (ACA/OA) bifurcations were assessed via a 191-gene data matrix expression profile by ATAC-PCR. We identified 15 genes whose expression is significantly altered during cerebral aneurysm formation, including major heparan sulfate proteoglycan, cathepsin B, hevin and β₄-integrin. We also confirmed protein expression of β₄-integrin in rat cerebral aneurysms by quantitative real-time PCR and immunohistochemistry. The ATAC-PCR revealed temporal changes in gene expression during the development of experimental cerebral aneurysms. The genes that were significantly changed in this study would be the candidates for future studies concerning the development of cerebral aneurysms.

Simvastatin Suppresses the Progression of Experimentally Induced Cerebral Aneurysms in Rats

The pathophysiology of cerebral aneurysms (CAs) is linked to chronic inflammation and degradation of extracellular matrix in vascular walls. Because statins have protective effects on various vascular diseases independent of their lipid-lowering effects, we investigated the effect of simvastatin on CA progression. CAs were induced in Sprague-Dawley rats with or without oral administration of simvastatin. The size and media thickness of CAs was evaluated 3 months after aneurysm induction. Expression of macrophage chemoattractant protein-1, vascular cell adhesion molecule-1, endothelial nitric oxide synthase, interleukin-1beta, inducible nitric oxide synthase, matrix metalloproteinase-2, and matrix metalloproteinase-9 in aneurysmal walls was examined by reverse transcriptase-polymerase chain reaction and immunohistochemistry. To examine whether simvastatin has a suppressive effect on preexisting CAs, simvastatin administration started at 1 month after aneurysm induction. Rats treated with simvastatin exhibited a significant increase in media thickness and a significant reduction in aneurysmal size compared with control rats. Treatment with simvastatin resulted in reduced expression of macrophage chemoattractant protein-1 and vascular cell adhesion molecule-1, increased expression of endothelial nitric oxide synthase, and reduced the number of macrophage infiltration. In quantitative polymerase chain reaction and immunohistochemistry, simvastatin significantly inhibited upregulated expression of interleukin-1beta, inducible nitric oxide synthase, matrix metalloproteinase-2, and matrix metalloproteinase-9 associated with CA progression. Gelatin zymography revealed decreased activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 in aneurysmal walls by simvastatin treatment. Simvastatin also effectively inhibited aneurysm enlargement and thinning of the media of preexisting CAs. Treatment with simvastatin suppresses the development of CAs by inhibiting inflammatory reactions in aneurysmal walls. Simvastatin also has a preventive effect on the progression of preexisting CAs. Simvastatin is a promising candidate of a novel medical treatment for the prevention of CA progression.

Role of TIMP-1 and TIMP-2 in the Progression of Cerebral Aneurysms

The degradation of extracellular matrix (ECM) is a hallmark of a cerebral aneurysm; however, little is known regarding the molecular mechanism leading to this change. Tissue inhibitor of matrix metalloproteinase (TIMP) regulates the ECM degradation in vascular walls by inhibiting the activity of matrix metalloproteinases (MMPs). We investigated the role of TIMPs in the progression of cerebral aneurysms in the present study. TIMP-1 and TIMP-2 expression was examined by immunohistochemistry and quantitative RT-PCR in experimentally-induced cerebral aneurysms in rats. The incidence of aneurysmal changes in TIMP-1(-/-) and TIMP-2(-/-) mice was compared with that in the wild-type mice. TIMP-1 and TIMP-2 were expressed mainly by smooth muscle cells in aneurysmal walls. Quantitative PCR showed an increase of TIMP-1 and TIMP-2 mRNA in the early stage of aneurysm progression (form 0 to 1 month) but not in the late stage (form 1 to 3 months), whereas mRNA expression of MMP-2 and MMP-9 dramatically increased in the late stage. In both TIMP-1(-/-) mice and TIMP-2(-/-) mice, aneurysm progression was promoted with the increased enzyme activity of MMPs. Our findings suggest that TIMP-1 and TIMP-2 have a protective role for the progression of cerebral aneurysms. There is an imbalance between MMPs and TIMPs in the late stage of cerebral aneurysm formation, which may be responsible for ECM degradation leading to the progression and rupture of cerebral aneurysms.

Macrophage-Derived Matrix Metalloproteinase-2 and -9 Promote the Progression of Cerebral Aneurysms in Rats

Mechanisms of initiation, progression and rupture of cerebral aneurysms have not yet been fully understood despite its clinical significance. Matrix metalloproteinases (MMPs) are a family of proteinases which are involved in the remodeling of vascular walls. In the present study, we investigated the significance of MMPs in the progression of cerebral aneurysms. Cerebral aneurysms were experimentally induced in 7-week-old male Sprague-Dawley rats. MMP-2 and MMP-9 expression was examined by immunohistochemistry and RT-PCR. Gelatinase activity in aneurysmal walls was assessed by in situ zymography. A selective inhibitor for MMP-2, -9 and -12, tolylsam, was used to examine the effect of inhibition of MMP-2 and MMP-9. Macrophages infiltrated in arterial walls of experimentally induced rat cerebral aneurysms and expressed MMP-2 and -9. Macrophage infiltration and MMP expression was increased with the progression of aneurysms. Gelatinase activity attributable to MMP-2 and MMP-9 increased in arterial walls of rat cerebral aneurysms. Furthermore, tolylsam reduced the ratio of advanced aneurysms in our rat model. These data suggest that macrophage-derived MMP-2 and -9 may play an important role in the progression of cerebral aneurysms. The findings of this study will shed a new light into the pathogenesis of cerebral aneurysms and highlight the importance of inflammatory response causing the degeneration of extracellular matrix in the process of this disease.

Impaired Progression of Cerebral Aneurysms in Interleukin-1β–Deficient Mice

Subarachnoid hemorrhage caused by cerebral aneurysm rupture remains a life-threatening emergency despite advances in treatment. However, the mechanisms underlying aneurysm initiation, progression, and rupture remain unclear. We developed a method to induce experimental cerebral aneurysms in rats, monkeys, and mice. Interleukin-1beta (IL-1beta) is a key inflammatory mediator, and it is thought to be a promising target for the treatment of inflammatory diseases. In the present study, we examined the role of IL-1beta in cerebral aneurysm development. Cerebral aneurysms were experimentally induced in 5-week-old male C57BL/6 mice, IL-1beta gene-deficient (IL-1beta-/-) mice, and age-matched control B10 mice (wild-type). Their cerebral arteries were dissected and examined histologically and immunohistochemically. IL-1beta was expressed in vascular media in mice at an early stage of aneurysmal models' cerebral arteries. No differences were seen in the rate of aneurysm development between IL-1beta-/- and wild-type mice, but the percentage of advanced aneurysm change was significantly larger in wild-type animals. Furthermore, in IL-1beta-/- mice, increased caspase-1 expression was seen compared with wild-type animals. Additionally, the number of apoptotic cells assessed by single-stranded DNA immunoreactivity and TUNEL was significantly reduced in IL-1beta-/- mice compared with wild-type animals. IL-1beta is important for the progression of cerebral aneurysms in a mouse model. Disruption of the IL-1beta gene results in the reduced incidence of mature experimental cerebral aneurysms.

Role of estrogen deficiency in the formation and progression of cerebral aneurysms. Part I: experimental study of the effect of oophorectomy in rats

Estrogen has been shown to play a central role in vascular biology. Although it may exert beneficial vascular effects, its role in the pathogenesis of cerebral aneurysms remains to be determined. To elucidate the role of hormones further, the authors examined the effects of bilateral oophorectomy on the formation and progression of cerebral aneurysms in rats. Forty-five female, 7-week-old Sprague-Dawley rats were divided into three equal groups. Group I consisted of intact rats (controls). To induce cerebral aneurysms, the animals in Groups II and III were subjected to ligation of the right common carotid and bilateral posterior renal arteries. One month later, the rats in Group II underwent bilateral oophorectomy. Three months after the experiment began all animals were killed and cerebral vascular corrosion casts were prepared and screened for cerebral aneurysms by using a scanning electron microscope. Plasma was used to determine the level of estradiol and the gelatinase activity. Hypertension developed in all rats except those in the control group. The estradiol level was significantly lower in Group II than in the other groups (p < 0.01). The incidence of cerebral aneurysm formation in Group II (60%) was three times higher than that in Group III (20%), and the mean size of aneurysms in Group II (76 +/- 27 microm, mean +/-standard deviation) was larger than that in Group III (28 +/- 4.6 microm) (p < 0.05). No aneurysm developed in control animals (Group I), and there was no significant difference in plasma gelatinase activity among the three groups. The cerebral aneurysm model was highly reproducible in rats. Bilateral oophorectomy increased the susceptibility of rats to aneurysm formation, indicating that hormones play a role in the pathogenesis of cerebral aneurysms.

Role of estrogen deficiency in the formation and progression of cerebral aneurysms. Part II: experimental study of the effects of hormone replacement therapy in rats

The increased incidence of cerebral aneurysms in postmenopausal women appears to be related to low levels of circulating estrogen. Using a rat model of aneurysm induction, the authors found that oophorectomy increased the incidence of experimental cerebral aneurysms (Part I in this issue). In the current study they examined the effects of hormone replacement therapy (HRT) on the formation of cerebral aneurysms in rats. Forty-five female Sprague-Dawley rats were divided into three equal groups. The animals in Groups A and B were subjected to a cerebral aneurysm induction procedure (renal hypertension and right common carotid artery ligation) followed 1 month later by bilateral oophorectomy. After an additional week the rats in Group A received 17beta estradiol continuous-release pellets. The rats in Group C served as controls. Three months after the aneurysm induction procedure, all the rats were killed and vascular corrosion casts of their cerebral arteries were prepared and checked for aneurysmal changes. Using a scanning electron microscope, the authors recorded aneurysmal changes as endothelial changes alone (Stage I), endothelial changes with intimal pad elevation (Stage II), and saccular aneurysm formation (Stage III). Aneurysmal changes (Stages I, II, and III) occurred in one third of rats that had undergone oophorectomy and were receiving HRT (Group A), compared with 87% of the rats that had undergone oophorectomy but did not receive HRT (Group B). Although most of the aneurysmal changes identified in Group A rats were limited to Stage I or II, most changes in Group B animals were identified as saccular dilation (Stage III). The findings demonstrated the significant protective role of estrogen against the formation and progression of cerebral aneurysms. It appears to be related to the beneficial effects of estrogen on the function and growth of endothelial cells, which play a major role in preserving the integrity of the vascular wall.

Disruption of gene for inducible nitric oxide synthase reduces progression of cerebral aneurysms.

The rupture of a cerebral aneurysm is a major cause of subarachnoid hemorrhage, but the mechanism of its development remains unclear. Inducible nitric oxide synthase (iNOS) is expressed in human and rat cerebral aneurysms, and aminoguanidine, a relatively selective inhibitor of iNOS, can decrease the number of the aneurysms in rats. In this study we applied our new mouse model of cerebral aneurysms to the iNOS gene knockout mice and observed experimental cerebral aneurysms in these animals to elucidate the role of iNOS in the process of cerebral aneurysm formation. Eight C57/Bl6 mice and 16 iNOS knockout mice received a cerebral aneurysm induction procedure. Four months after the operation, the mice were killed, their cerebral arteries were dissected, and the region of the bifurcation of the anterior cerebral artery/olfactory artery was examined histologically and immunohistochemically. No significant difference was seen in the incidence of cerebral aneurysms between iNOS+/+ and iNOS-/- mice. However, the size of advanced cerebral aneurysms and the number of apoptotic smooth muscle cells were significantly greater in iNOS+/+ mice than in iNOS-/- mice. Inducible NOS is not necessary for the initiation of cerebral aneurysm. However, the results of this study suggest that regulation of iNOS may have therapeutic potential in the prevention of the progression of cerebral aneurysms.

Gene expression profile of the intima and media of experimentally induced cerebral aneurysms in rats by laser-microdissection and microarray techniques

Cerebral aneurysm is a common disease with a high prevalence and can cause a catastrophic subarachnoid hemorrhage. To elucidate the molecular mechanism of the formation and progression of cerebral aneurysms, gene expression profiling was performed in experimentally induced rat cerebral aneurysms. The intima and media of cerebral arterial walls in rats with or without aneurysm induction were dissected respectively by a laser-microdissection technique. Changes in gene expression in the intima and media of aneurysmal walls were analyzed using Agilent Rat Oligo Microarrays, followed by a specific pathway analysis using GeneSpring software. Of the 41,012 genes examined, 633 were differentially expressed between a normal cerebral artery and a cerebral aneurysm in the intima, with 395 showing increased expression and 238 showing decreased expression. In the media, 1344 were differentially expressed, with 928 showing increased expression and 416 showing decreased expression. Specific pathway analysis revealed that increased gene expression was associated with proteinase, reactive oxygen species, growth factor, chemokine, complement, adhesion molecule and apoptosis in both the intima and the media of aneurysmal walls. Some genes showed an opposite expression pattern between the intima and the media indicating a different role between endothelial cells and vascular smooth muscle cells in cerebral aneurysm formation and progression. These data suggest that cerebral aneurysmal formation and progression are closely related to vascular inflammation, degeneration of extracellular matrix and apoptosis.

Apoptosis of medial smooth muscle cells in the development of saccular cerebral aneurysms in rats.

Using an animal model, we examined the role of apoptosis in the disappearance of medial smooth muscle cells (SMCs) during the development and growth of cerebral aneurysms. Various degrees of cerebral aneurysms were induced in the right anterior cerebral artery-olfactory artery bifurcations in 65 Sprague-Dawley rats with ligation of the left common carotid artery and renal hypertension. We performed in situ end labeling of fragmented DNA with the lesions in 45 rats and electron microscopic study in the other 20 rats. With in situ end labeling of fragmented DNA, 4+/-3 apoptotic medial SMCs were detected in 35 of the 45 bifurcations. Apoptotic SMCs appeared in the medial layer in the "preaneurysm" group, the site speculated to show an aneurysmal change in the near future (6+/-3), and in the media in the "early aneurysm" group, which showed characteristics such as a small depression (5+/-3). In the "progressive aneurysm" group, they appeared more frequently at the aneurysmal neck (3+/-2) than the dome (1+/-1). By electron microscopic study, shrunken medial SMCs exhibiting morphological apoptotic changes such as chromatin condensation and fragmentation of the cytoplasm and nucleus were observed in the preaneurysm and early aneurysm groups and at the neck portion in the progressive aneurysm group. In the aneurysmal dome, SMCs showed late characteristics of apoptosis such as more advanced nuclear and cytoplasmic condensation and formation of apoptotic bodies. The present findings indicate that there is an association between apoptosis of medial SMCs and the formation of saccular cerebral aneurysms.

Immunohistochemical Alterations of Fibronectin During the Formation and Proliferative Repair of Experimental Cerebral Aneurysms in Rats

To determine whether distributional changes of fibronectin, a factor promoting wound healing, occur during the formation and repair of cerebral saccular aneurysms, we performed immunohistochemical analyses in experimental aneurysms. Cerebral aneurysms were induced in rats by both the ligation of the unilateral common carotid artery and induced hypertension. Intimal proliferation in aneurysmal walls was induced by the ligation of the preserved common carotid artery 3 months after the first operation. The distribution of fibronectin was examined by immunohistochemistry in anterior cerebral artery-olfactory artery bifurcations under the following three conditions: normal bifurcations in control rats, early aneurysmal lesions during the aneurysm induction, and aneurysmal lesions with intimal proliferation. Furthermore, the immunohistochemical distributions of type I and IV collagens were examined to evaluate the specificity of fibronectin immunoreactivity. In the normal bifurcations, fibronectin was positive in the subintimal space, the surrounding area of the medial smooth muscle cells, and the adventitial fibrous tissue. In early aneurysmal lesions, linear staining of fibronectin and type I and IV collagens in the subendothelial space disappeared with the loss of the internal elastic lamina. In the intimal proliferation of early aneurysmal lesions, fibronectin was strongly immunostained in the subendothelial space and diffusely immunostained in the widened extracellular space surrounding proliferated cells. In contrast, the stainings of type I and IV collagens were sparse or negative. Although the present findings regarding dynamic changes of fibronectin distribution do not prove any causality in the process of aneurysm formation and repair, these immunohistochemical changes may constitute the crucial sequela of intimal endothelial damage and its subsequent recovery in cerebral aneurysms.

Basic fibroblast growth factor may repair experimental cerebral aneurysms in rats.

To determine whether basic fibroblast growth factor (FGF) can induce proliferative response of endothelial cells and/or smooth muscle cells in aneurysmal lesions, we investigated the effect of the intravenous administration of basic FGF on experimental cerebral aneurysms. Cerebral aneurysms were induced in rats by ligation of the unilateral common carotid artery, producing hypertension. Three months later, basic FGF was intravenously injected in two groups of randomly divided rats on days 1, 3, and 5 at two different doses (low dose: 2 micrograms/100 g body wt per day; high dose: 5 micrograms/100 g body wt per day). In a control group, normal saline was similarly injected. The junctions of the anterior cerebral artery (ACA) and the olfactory artery (OA) were examined with a light microscope. Aneurysmal changes were defined as the lesions with discontinuity of the internal elastic lamina in more than half of the outward dilated wall. Depending on whether the smooth muscle cell layer was present in the whole wall, the lesions were divided into two stages: early aneurysmal lesion (whole area) and saccular aneurysm (not totally preserved). The control and the low-dose groups presented no obvious intimal thickening in the intact ACA-OA junctions of both nonligated and ligated sides as well as in the aneurysmal changes. In contrast, in the high-dose group, various degrees of intimal thickening in the wall were detected in 7 of 15 early aneurysmal lesions (P = .019, Fisher's exact test). Immunohistochemistry showed the proliferated cells to be smooth muscle cells. These results demonstrate that exogenous basic FGF induces the proliferative response of smooth muscle cells in aneurysmal lesions in rats.