Mechanisms of formation and growth of intracranial aneurysms are poorly understood. To investigate the pathophysiology of intracranial aneurysms, an animal model of intracranial aneurysm yielding a high incidence of large aneurysm formation within a short incubation period is needed. We combined two well-known clinical factors associated with human intracranial aneurysms, hypertension and the degeneration of elastic lamina, to induce intracranial aneurysm formation in mice. Roles of matrix metalloproteinases (MMPs) in this model were investigated using doxycycline, a broad-spectrum MMP inhibitor, and MMP knockout mice. Hypertension was induced by continuous infusion of angiotensin II for 2 weeks. The disruption of elastic lamina was achieved by a single stereotaxic injection of elastase into the cerebrospinal fluid at the right basal cistern. A total of 77% of the mice that received 35 milliunits of elastase and 1000 ng/kg per minute of angiotensin II developed intracranial aneurysms in 2 weeks. There were dose-dependent effects of elastase and angiotensin II on the incidence of aneurysms. Histologically, intracranial aneurysms observed in this model closely resembled human intracranial aneurysms. Doxycycline, a broad-spectrum MMP inhibitor, reduced the incidence of aneurysm to 10%. MMP-9 knockout mice, but not MMP-2 knockout mice, had reduced the incidence of intracranial aneurysms. In summary, a stereotaxic injection of elastase into the basal cistern in hypertensive mice resulted in intracranial aneurysms that closely resembled human intracranial aneurysms. The intracranial aneurysm formation in this model appeared to depend on MMP activation.
Intracranial aneurysms are considered to be common among the general population. Subarachnoid hemorrhage from ruptured intracranial aneurysms results in catastrophic consequences causing severe morbidity and high mortality.1 Despite recent advances in diagnosis and treatment, the mechanisms for the formation, growth, and subsequent rupture of intracranial aneurysms are not yet well understood.
Clinically, systemic hypertension is associated with intracranial aneurysm formation and subarachnoid hemorrhage from aneurysmal rupture.2–4 However, a causal relationship between hypertension and intracranial aneurysm formation or subarachnoid hemorrhage has not been fully established. Histologically, degeneration and disruption of the elastic lamina are key characteristics of human intracranial aneurysm.2,5,6 Degeneration or disruption of elastic lamina may be attributed to the normal aging process or damages caused by hemodynamic stresses.7 Such changes in the elastic lamina have often been considered as preaneurysmal changes that eventually lead to the maturation of aneurysms.8,9 Elastase-induced fragmentation of elastic lamina has been successfully used to induce aneurysms in the carotid artery10,11 and aorta12 in animals. In these models, because of the ease of surgical or endovascular access of these blood vessels, elastase was applied intraluminally to induce degeneration of elastic lamina.
In this study, we present a new mouse model of intracranial aneurysms. To induce intracranial aneurysm formation, we combined hypertension and the degeneration of elastic lamina, two well-known clinical factors associated with human intracranial aneurysms. Hypertension was induced by continuous infusion of angiotensin II; degradation of elastic lamina was induced by a single injection of elastase into the cerebrospinal fluid using a stereotaxic method. Intracranial aneurysms in this model histologically mimic human intracranial aneurysms. In addition, roles of matrix metalloproteinases (MMPs) in this model were investigated using the MMP inhibition by doxycycline, a broad-spectrum MMP inhibitor, and MMP knockout mice.
Methods
Detailed descriptions of the methods are provided in online Data Supplement at http://hyper.ahajournals.org.
To test whether the combination treatment of single elastase injection and pharmacologically induced hypertension can cause the formation of intracranial aneurysms in mice, we treated C57BL/6J mice (8 to 10 weeks old) with a single stereotaxic injection of elastase at the right basal cistern, immediately followed by induction of systemic hypertension with continuous injection of angiotensin II. After 2 weeks, we euthanized the mice and perfused the animals with a bromophenol blue dye and gelatin mixture.
Our preliminary study indicated a wide variation in vessel diameter for the Circle of Willis and its major branches in mice. To have a conservative and consistent control for our experiment, we used the diameter of the basilar artery, one of the larger vessels in the brain circulation with relatively little variation, as a reference blood vessel. Aneurysms were operationally defined as a localized outward bulging of the vascular wall of which the diameter is >150% of the basilar artery diameter. The Circle of Willis or its major primary branches were assessed by 2 investigators in a blinded manner.
To assess the relationship between the elastase dose and the incidence of aneurysms, 4 groups of mice were prepared: group 1 used 35.0 milliunits of heat-inactivated elastase; group 2 used PBS (n=10); group 3 used 3.5 milliunits of elastase (n=10); group 4 used 17.5 milliunits of elastase (n=20); and group 5 used 35 milliunits of elastase (n=44). Angiotensin II was administered at 1000 ng/kg per minute for 2 weeks in all of the groups.
To assess the relationship between the angiotensin II concentration and the incidence of aneurysms, 3 groups of mice were prepared: group 1 used continuous infusion of PBS via osmotic pump (n=10); group 2 used 500 ng/kg per minute of angiotensin II (n=10); and group 3 used 1000 ng/kg per minute of angiotensin II (n=44). This group (group 3) is the same as group 5 in the above-mentioned experiment testing the dose dependency of elastase effects. Thirty-five milliunits of elastase were administered to all of the groups.
Doxycycline Treatment and MMP Knockout Mice
To assess the effects of doxycycline, a broad-spectrum MMP inhibitor, on the incidence of aneurysms, 10 mice received the doxycycline through drinking water (40 mg/kg per day). Doxycycline treatment was started 3 days before the elastase injection and continued for 17 days (3 days+2 weeks). Previously, this dose administered through drinking water was used to successfully suppress MMPs involved in various disease models in mice.13–15 For the control group, 10 mice received drinking water without doxycycline. These mice are different from the mice used in the dose-response studies describe above. To assess of roles of MMP-9 and MMP-2 specifically, we used MMP-9 knockout mice and MMP-2 knockout mice (n=10 in each group).16 All of the mice received a single injection of 35 milliunits of elastase and angiotensin II (1000 ng/kg per minute).
Statistical Methods
For continuous variables, we used ANOVA followed by Fisher least significant difference test, which was performed as a posthoc analysis. When repeated measurements were performed, ANOVA with a repeated measurement design was used. For the incidence, we used the χ2 test. Data are presented as mean±SD. Statistical significance was taken at P<0.05.
Results
Formation of Intracranial Aneurysm by a Combination of a Single Stereotaxic Injection of Elastase Into the Basal Cistern and Angiotensin II-Induced Hypertension
Among the mice that received a single stereotaxic injection of elastase at 35 milliunits into the basal cistern and a continuous SC infusion of angiotensin II at 1000 ng/kg per minute, 77% of the mice (34 of 44) were found to have developed intracranial aneurysm formations along the Circle of Willis or its major branches.
Figure 1 shows a schematic view of normal brain vasculature (Figure 1A), normal cerebral arteries from a control brain (Figure 1B), and representative intracranial aneurysms (Figure 1C through 1E). To visualize the Circle of Willis and its major branches, mice were perfused with blue dye (bromophenol blue). In the mice treated with angiotensin II and elastase, large intracranial aneurysm formations were found mostly along the right half of the Circle of Willis and its major branches (Figure 1C through 1E). Most of the aneurysms were >500 μm, ≈3 to 5 times larger than their parent arteries. A mouse that died at day 12 had fresh blood clots along the right middle cerebral artery (Figure 1E), revealing subarachnoid hemorrhage. There was a larger aneurysm formation inside the blood clots of subarachnoid hemorrhage (Figure 1E).
Figure 1. Representative intracranial aneurysms. A, A schematic view of normal cerebral arteries. B, Normal cerebral arteries including the Circle of Willis, and its major branches are stained in blue. C through E, Base of the brain with intracranial aneurysm formations. Black arrows indicate intracranial aneurysm formations. Large intracranial aneurysm formations were found mostly along the right half of the Circle of Willis and its major branches. C, Intracranial aneurysm at the right posterior communicating artery. D, Intracranial aneurysm at the right middle cerebral artery. E, A mouse that died at day 12 had fresh blood clots along the right middle cerebral artery, revealing subarachnoid hemorrhage. Inside the blood clots of subarachnoid hemorrhage, there was a larger aneurysm formation. F, Locations of intracranial aneurysms found in the mice that received 35 milliunits of elastase and 1000 ng/kg per minute of angiotensin II (n=44). The majority of the intracranial aneurysms were located ipsilaterally to the elastase injection site. Small numbers of intracranial aneurysms were found on the contralateral side of the injection site, as well as on the branches of the basilar artery.
Figure 1F shows the locations of intracranial aneurysms in mice that received a single stereotaxic injection of 35 milliunits of elastase and a continuous infusion of angiotensin II at 1000 ng/kg per minute. The majority of the intracranial aneurysms were located ipsilaterally to the elastase injection site. This was consistent with the bromophenol blue dye distribution found in the test dye injection. Small numbers of intracranial aneurysms were found on the contralateral side of the injection site, as well as on the branches of the basilar artery.
Continuous infusion of angiotensin II successfully induced systemic hypertension. At 1 week and 2 weeks after the initiation of continuous infusion of angiotensin II at 1000 ng/kg per minute, the blood pressure was significantly higher than the baseline (109±11 versus 142±37 versus 140±30 mm Hg; P<0.05; Figure 2C).
Figure 2. Dose-dependent effects of angiotensin II and elastase on the incidence of intracranial aneurysms. A, Dose-dependent relationship between the incidence of intracranial aneurysms and the concentration of elastase. B, Dose-dependent relationship between the incidence of aneurysms and the concentration of angiotensin II. C, Systolic blood pressure in mice that received different doses of angiotensin II. There was a dose-dependent relationship between angiotensin II and systolic blood pressure. *P<0.05 vs the group that received PBS infusion instead of angiotensin I. #P<0.05 vs the baseline value in each group.
To investigate the kinetics of elastase activity after a single injection of elastase into the cerebrospinal fluid space, we analyzed elastase activity in the cerebrospinal fluid after elastase injection. Elastase activity levels were under the detection levels (<0.005 U/mL) at the baseline or after sham injection. At 30 minutes or at 6 hours after the injection of 35 milliunits of elastase (n=3 in each group), elastase levels were under the detection level. As a next step, we injected 350 milliunits of elastase, a dose 10 times higher than what was actually used to induce aneurysms. At 30 minutes after injecting 350 milliunits of elastase, the elastase levels were 0.025±0.003 U/mL (n=3). At 6 hours after injecting 350 milliunits of elastase, the elastase activity levels returned to levels under the detection level (n=3). These data indicate that the elastase activity after a single injection of elastase was relatively short.
Dose-Dependent Effects of Elastase and Angiotensin II on the Incidence of Intracranial Aneurysms
Figure 2A shows a dose-dependent relationship between the incidence of intracranial aneurysm and the concentration of elastase. The incidence of aneurysms in groups that received 35 milliunits of heat-inactivated elastase, as well as 0.0 (PBS injection), 3.5, 17.0, and 35.0 milliunits of elastase, were 0%, 0%, 10%, 30%, and 77%, respectively.
Figure 2B shows a dose-dependent relationship between the incidence of intracranial aneurysms and the concentration of angiotensin II. The total incidence of aneurysms in groups that received 0 (PBS infusion), 500, and 1000 ng/kg per minute of angiotensin II were 0%, 20%, and 77%, respectively. The blood pressures for groups that received 0 (PBS), 500, or 1000 ng/kg per minute of angiotensin II were 111.0±6.7, 127.0±18.8, and 142.0±37.0 mm Hg, respectively, at 1 week (P<0.05; Figure 2C), showing a dose-dependent effect of angiotensin II on the blood pressure.
Histological Assessment of Intracranial Aneurysm Tissues
Histological assessment of intracranial aneurysm tissues from this model showed aneurysm formations with varying degrees of structural abnormalities that were similar to those reported in human intracranial aneurysms.2 Generally, intracranial aneurysms in this model had a vascular wall with thick segments. Figure 3 shows a normal basilar artery (Figure 3A and 3C) and a representative intracranial aneurysm (Figure 3B and 3D).
Figure 3. Histological assessment of intracranial aneurysms in this model. In the normal cerebral artery from the control mouse (A and C), there were 2 to 3 layers of smooth muscle cells (A2) and a single, thin continuous layer of endothelial cells (A3). Fibroblasts were very scarce (A4). Elastica van Gieson and trichrome (C1 and C2) stainings showed 1 layer of elastic lamina. In an intracranial aneurysm (B and D), the thin vascular wall showed intact endothelial and smooth muscle cell layers, whereas the thick vascular wall showed discontinued endothelial cell layers and scattered, faint, smooth muscle staining (B2 and B3). Elastica van Gieson and trichrome stainings revealed severely disorganized elastic lamina in both thin and thick portions of the artery (D and E). Bar: 100 μm.
In the normal cerebral artery from control mouse (Figure 3A and 3C), there were 2 to 3 layers of smooth muscle cells (Figure 3A2) and a single, thin continuous layer of endothelial cells (Figure 3A3). Fibroblasts were very scarce (Figure 3A4). Elastica van Gieson and trichrome (Figure 3C1 and 3C2) staining showed 1 layer of elastic lamina, as described previously.2 In an intracranial aneurysm (Figure 3B and 3D), the thin vascular wall showed intact endothelial and smooth muscle cell layers, whereas the thick vascular wall showed discontinued endothelial cell layers and scattered, faint smooth muscle staining (Figure 3B2 and 3B3). Elastica van Gieson and trichrome stainings revealed severely disorganized elastic lamina in both thin and thick portions of the artery (Figure 3D and 3E).
Potential roles of inflammation in the growth of intracranial aneurysms (both ruptured and unruptured) have been suggested by observational studies analyzing the presence of inflammatory cells or inflammatory markers in human intracranial aneurysm tissues and serum samples.6,7,17,18 Therefore, we investigated the presence of inflammatory cells in our intracranial aneurysm samples. Figure 4 shows a normal cerebral artery and a representative intracranial aneurysm.
Figure 4. Panleukocyte (CD45), macrophage (CD68), and T-lymphocyte (CD4) stainings for a normal cerebral artery from the control mouse (A) and a representative intracranial aneurysm (B). A normal basilar artery from the control mouse (A1, A2, and A3) showed an absence of inflammatory cells. In the intracranial aneurysm tissue (B), leukocytes (CD45-positive cells) were present throughout the vascular wall (B1 and B4). Distribution of macrophages (CD68-positive cells) generally overlapped with the leukocyte distribution (B2 and B5). Small numbers of CD4-positive T lymphocytes were present in the thin-wall portion of the aneurysm. However, CD4-positive T lymphocytes were not detected in the thick aneurysm wall (B3 and B6). Bar: 100 μm.
A normal basilar artery from the control mouse (Figure 4A) showed an absence of inflammatory cells. In the intracranial aneurysm tissue (Figure 4B), leukocytes (CD45-positive cells) were present throughout the vascular wall (Figure 4B1). Distribution of macrophages (CD68-positive cells) generally overlapped with the leukocyte distribution (Figure 4B2). Small numbers of CD4-positive T lymphocytes were present in the thin-wall portion of the aneurysm. However, CD4-positive T lymphocytes were not detected in the thick aneurysm wall (Figure 4B4).
Potential Roles of MMPs in the Formation of Intracranial Aneurysms
The presence of inflammatory cells in intracranial aneurysms tissues suggests potential roles of proteinases produced by inflammatory cells in the formation of intracranial aneurysms.7,17 Studies showed elevation of serum elastase and collagenase in patients with intracranial aneurysms,19–22 and MMP-9 and MMP-2 were detected in human intracranial aneurysm tissues.23 To test potential contributions from MMPs to the formation of intracranial aneurysms in this model, 3 lines of experiments were performed. First, MMP activity in intracranial aneurysm tissues was assessed using in situ zymography. Second, we treated the mice with doxycycline, a broad-spectrum MMP inhibitor. Third, intracranial aneurysm formation was assessed in MMP-9 and MMP-2 knockout mice.
Figure 5A through 5F shows in situ zymography and hematoxylin/eosin stainings of a normal brain vasculature and a representative intracranial aneurysm in this model. Although the normal brain vasculature lacked any appreciable gelatinase activity (Figure 5B), intracranial aneurysms showed intense fluorescence, indicating robust gelatinase activity (Figure 5A). Pretreatment of the tissues with 1,10-phenanthroline monohydrate (MMP inhibitor) abolished the gelatinase activity (Figure 5C), showing that the gelatinase activity observed in the intracranial aneurysm tissues was from MMPs.
Figure 5. Roles of MMPs in intracranial aneurysm formation. In situ zymography with or without an MMP inhibitor was performed on a normal cerebral artery (B) and a representative intracranial aneurysm in this model (A). Although the normal brain vasculature lacked any appreciable gelatinase activity (B), intracranial aneurysms showed intense fluorescence, indicating robust gelatinase activity (A). Pretreatment of the tissues with 1,10-phenanthroline monohydrate (MMP inhibitor) abolished the gelatinase activity (C), showing that the gelatinase activity observed in the intracranial aneurysm tissues was from MMPs. Incidence of intracranial aneurysms in 4 groups of mice is presented in G. The incidence of intracranial aneurysms in the control group (wild type) was 70%, consistent with the results from the dose-dependence studies described above. The doxycycline treatment reduced the incidence of intracranial aneurysms to 10% (P<0.05). The incidence of intracranial aneurysms was reduced to 40% in MMP-9 knockout mice compared with the wild-type mice (P<0.05). However, there were no differences in the incidences of aneurysms between the wild-type mice and MMP-2 knockout mice. KO indicates knockout mice.
Figure 5G shows the incidence of intracranial aneurysms in the control group (wild type), doxycycline-treated group (wild type), MMP-9 knockout mice, and MMP-2 knockout mice. All of the groups received the elastase injection and angiotensin II infusion. The incidence of intracranial aneurysms in the control group (wild type) was 70%, consistent with the results from the dose-dependence studies described above. The doxycycline treatment reduced the incidence of intracranial aneurysms to 10% (P<0.05), indicating potential roles for MMPs in the formation of aneurysms in this model. Similar results were reported previously using a different model of intracranial aneurysms in rats.24 The incidence of intracranial aneurysms was reduced to 40% in MMP-9 knockout mice compared with the wild-type mice (P<0.05). However, there was no difference in the incidence of aneurysms between the wild-type mice and MMP-2 knockout mice (70% versus 60%).
Discussion
In this study, we showed that the combination of hypertension and a single stereotaxic injection of elastase into the cerebrospinal fluid at the basal cistern resulted in the formation of intracranial aneurysms that recapitulated key features of human intracranial aneurysms. The single stereotaxic injection of elastase was used to induce disorganization or disruption of elastic lamina, a structural change that is considered to be preaneurysmal change.10,11,25 To induce systemic hypertension, we used continuous infusion of angiotensin II via a subcutaneously implanted osmotic pump. This is a well-established and easily reproducible method of inducing hypertension in mice.26,27 Although neither systemic hypertension nor degeneration of elastic lamina alone could cause intracranial aneurysms in our model, the combination of both factors resulted in large aneurysm formation. These findings suggest the synergistic effects of these 2 factors in the formation of intracranial aneurysms in this model. In addition, we were able to show dose-dependent effects of angiotensin II and elastase on the formation of aneurysms, further supporting the causative roles of hypertension and degeneration of elastic lamina in aneurysm formation in this model.
Aneurysms formed in this model were generally large and easily distinguishable from the normal arteries without using histological assessment. Aneurysm formation in this model can serve as a simple and easily interpretable outcome for future studies that use various inhibitors, knockout mice, or transgenic mice to test the roles of specific molecules and pathways in the pathophysiology of intracranial aneurysms.
Histologically, intracranial aneurysms observed in this model closely resembled human intracranial aneurysms.2 The intracranial aneurysms in this model showed thin and thick vascular walls with partial loss of smooth muscle cells; elastic laminas showed various degenerative changes ranging from a partial disruption to a complete loss. In addition, the infiltration of leukocytes, especially macrophages, into the aneurysm wall was clearly present in our model, consistent with the observational studies that analyzed human intracranial aneurysm tissues and serum samples.6,17
In this model, intracranial aneurysms formed within 2 weeks. Such a short incubation time for this model’s intracranial aneurysm formation would allow practical and feasible screening of various molecular targets. However, it should be cautioned that aneurysm formation and maturation are believed to require a much longer time frame in humans. The relatively shorter time span of aneurysm formation in this model may be attributable to the higher magnitude of physiological insults (hypertension and elastase-induced inflammation) generated to create the model. In animal models, inciting factors that lead to the disease often need to be exaggerated or accentuated to achieve a severe disease phenotype at high incidence within a practical time frame. However, by doing so, the model may fail to recapitulate or skip events that play key roles in the human disease.
Although angiotensin II-induced hypertension is a well-established, well-characterized model of hypertension in mice,26,27 the effects of angiotensin II are diverse. Angiotensin II can exert various effects on the vasculature in addition to its hypertensive effect, including induction of reactive oxygen species and promotion of inflammation.28–30 Such nonhemodynamic effects of angiotensin II may have contributed to intracranial aneurysm formation in this model. In abdominal aortic aneurysms induced by angiotensin II in apolipoprotein E knockout mice, the nonhemodynamic effects appeared to be major causative factors.31,32 Additional studies are needed to elucidate relative contributions from nonhemodynamic effects and hypertensive effects of angiotensin II in this model.
The potential roles of proteinases, especially MMPs, in the formation of intracranial aneurysms have been suggested by observational, genetic, and experimental studies.19–23,33 In our study, we detected increased MMP activity in intracranial aneurysm tissues, and the treatment with doxycycline, a broad-spectrum MMP inhibitor, dramatically reduced the incidence of intracranial aneurysms. Interestingly, the incidence of aneurysms was significantly reduced in MMP-9 knockout mice but not in MMP-2 knockout mice. MMP-9 is known to be produced by inflammatory cells, especially macrophages, and its expression is upregulated in vascular diseases, including abdominal aortic aneurysms.34,35 On the other hand, MMP-2 is constitutively expressed by fibroblasts and smooth muscle cells in vascular tissues. Hemodynamic stress induced by systemic hypertension can trigger an inflammatory process by activating endothelial and inflammatory cells and upregulating leukocyte adhesion molecules.36 In addition, the degeneration of elastic lamina by elastase in this model can trigger vascular inflammation. MMP-9 produced by inflammatory cells may destabilize the vascular wall and facilitate excessive vascular remodeling, causing aneurysm formation. The constitutive nature of MMP-2 expression may suggest that MMP-2 is not critical for dynamic vascular remodeling processes that may be occurring during aneurysm formation. Alternatively, unknown developmental compensation may have masked effects of the lack of MMP-2 on intracranial aneurysm formation in this model.
It should be noted that the greater reduction in the incidence of aneurysms in doxycycline-treated mice compared with MMP-9 knockout mice may suggest potential contributions from other MMPs to aneurysm formation in this model. In addition, doxycycline has other effects in addition to inhibiting MMPs, including the modulations of various aspects of inflammation.37,38 These additional effects of doxycycline may have diminished or masked its MMP-inhibitory effects. Nevertheless, these data showed the feasibility of using this model to study underlying mechanisms for intracranial aneurysm formation.
Hashimoto and colleagues5,25,39,40 at Kyoto University pioneered the development of an elaborate intracranial aneurysm model that combines 3 surgical and pharmacological manipulations. They combined the following 3 parts: (1) renovascular hypertension induced by ligation of posterior branches of the bilateral renal arteries and loading with a high-salt diet; (2) continuous administration of a lathyrogen, β-aminopropionitrile; and (3) unilateral ligation of a common carotid artery. Animals in this model developed preaneurysmal changes or microscopic intracranial aneurysm formations at the olfactory artery-anterior cerebral artery bifurcation after 3 to 4 months. Not only did their model and our model share the histological characteristics of aneurysms, but both models also showed that pharmacological inhibitions of MMPs result in the reduction of intracranial aneurysm formation.24 Although the initial events that lead to aneurysm formation in these 2 model may be different, both models may be sharing common downstream pathways, including MMP activation, that lead to the same end phenotype: aneurysm formation.
Perspectives
We established a new mouse model of intracranial aneurysm that recapitulates key characteristics of human intracranial aneurysms. In this model, the combination of hypertension and the degeneration of elastic lamina induced by a single stereotaxic injection of elastase into the cerebrospinal fluid resulted in large aneurysm formations with the incidence as high as 77%. The aneurysm formation in this model appeared to depend on MMP activity. This model can be used to study molecular mechanisms that lead to aneurysm formation and growth.
Acknowledgments
We thank Dr William L. Young for providing mentoring and insightful suggestion and Mark Weinstein for his skillful technical assistance.
Sources of Funding
This study was funded by National Institutes of Health grants R01NS055876 (to T.H.) and P01NS044155 (to T.H.) and American Heart Association Grant-in-Aid 0755102Y (to T.H.).
Disclosures
None.
Footnote
Y.N. and T.-L.T. contributed equally to this study.
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On the cover: Immunofluorescene single or double labeling of transient receptor potential vanilloid 1 (TRPV1) and endothelin B (ETB) receptors in the renal pelvis of wild-type or TRPV1–/– mice. TRPV1 was labeled with fluorescein isothiocyanate (green fluorescence, arrow) in the left column, while the ETB receptors were labeled with Cy3 (red fluorescence, arrows) in the middle column. The overlay of TRPV1 and ETB is shown in the right column (yellow staining, arrows). The controls by preabsorption of primary antibodies are negative (not shown). (See page 1298.)
From the Department of Anesthesia and Perioperative Care (Y.N., T.-L.T., C.K., M.K., Y.K., T.H.) and Center for Cerebrovascular Research (Y.N., T.-L.T., C.K., M.K., Y.K., T.H.), University of California, San Francisco, San Francisco, Calif.
From the Department of Anesthesia and Perioperative Care (Y.N., T.-L.T., C.K., M.K., Y.K., T.H.) and Center for Cerebrovascular Research (Y.N., T.-L.T., C.K., M.K., Y.K., T.H.), University of California, San Francisco, San Francisco, Calif.
From the Department of Anesthesia and Perioperative Care (Y.N., T.-L.T., C.K., M.K., Y.K., T.H.) and Center for Cerebrovascular Research (Y.N., T.-L.T., C.K., M.K., Y.K., T.H.), University of California, San Francisco, San Francisco, Calif.
From the Department of Anesthesia and Perioperative Care (Y.N., T.-L.T., C.K., M.K., Y.K., T.H.) and Center for Cerebrovascular Research (Y.N., T.-L.T., C.K., M.K., Y.K., T.H.), University of California, San Francisco, San Francisco, Calif.
From the Department of Anesthesia and Perioperative Care (Y.N., T.-L.T., C.K., M.K., Y.K., T.H.) and Center for Cerebrovascular Research (Y.N., T.-L.T., C.K., M.K., Y.K., T.H.), University of California, San Francisco, San Francisco, Calif.
From the Department of Anesthesia and Perioperative Care (Y.N., T.-L.T., C.K., M.K., Y.K., T.H.) and Center for Cerebrovascular Research (Y.N., T.-L.T., C.K., M.K., Y.K., T.H.), University of California, San Francisco, San Francisco, Calif.
Correspondence to Tomoki Hashimoto, University of California, San Francisco, 1001 Potrero Ave, No. 3C-38, San Francisco, CA 94110. E-mail [email protected]
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Multiomics integrated analysis and experimental validation identify TLR4 and ALOX5 as oxidative stress-related biomarkers in intracranial aneurysms, Journal of Neuroinflammation, 21, 1, (2024).https://doi.org/10.1186/s12974-024-03226-0
Current Mouse Models of Intracranial Aneurysms: Analysis of Pharmacological Agents Used to Induce Aneurysms and Their Impact on Translational Research, Journal of the American Heart Association, 13, 3, (2024)./doi/10.1161/JAHA.123.031811
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Elastase-Induced Intracranial Aneurysms in Hypertensive Mice
Hypertension
Vol. 54
No. 6
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