Successful serial imaging of the mouse cerebral arteries using conventional 3-T magnetic resonance imaging

Abstract

Serial imaging studies can be useful in characterizing the pathologic and physiologic remodeling of cerebral arteries in various mouse models. We tested the feasibility of using a readily available, conventional 3-T magnetic resonance imaging (MRI) to serially image cerebrovascular remodeling in mice. We utilized a mouse model of intracranial aneurysm as a mouse model of the dynamic, pathologic remodeling of cerebral arteries. Aneurysms were induced by hypertension and a single elastase injection into the cerebrospinal fluid. For the mouse cerebrovascular imaging, we used a conventional 3-T MRI system and a 40-mm saddle coil. We used non-enhanced magnetic resonance angiography (MRA) to detect intracranial aneurysm formation and T2-weighted imaging to detect aneurysmal subarachnoid hemorrhage. A serial MRI was conducted every 2 to 3 days. MRI detection of aneurysm formation and subarachnoid hemorrhage was compared against the postmortem inspection of the brain that was perfused with dye. The imaging times for the MRA and T2-weighted imaging were 3.7±0.5 minutes and 4.8±0.0 minutes, respectively. All aneurysms and subarachnoid hemorrhages were correctly identified by two masked observers on MRI. This MRI-based serial imaging technique was useful in detecting intracranial aneurysm formation and subarachnoid hemorrhage in mice.

Introduction

Cerebral arteries undergo remodeling in response to physiologic and pathologic stimuli, resulting in changes in the diameter of cerebral arteries.¹ There are a number of mouse models of the physiologic or pathologic remodeling of cerebral arteries.^{2, 3, 4, 5} To study the remodeling of cerebral arteries, a noninvasive, serial imaging technique that can visualize the cerebral arteries is an extremely useful tool.

We report on the feasibility of using a readily available, conventional 3-T clinical magnetic resonance imaging (MRI) system to serially image cerebral arteries in mice. As a test case, we utilized a mouse model of an intracranial aneurysm that shows dynamic changes in the cerebral arteries.^{2, 6, 7} In this model, intracranial aneurysms were induced by a combination of systemic hypertension and a single injection of elastase into the cerebrospinal fluid.² The aneurysms develop within 7 days after the elastase injection. And they were approximately 500 μm and found in the Circle of Willis and its major branches.² Interestingly, aneurysmal rupture in this model results in subarachnoid hemorrhage with neurologic symptoms including a reduced motor activity and paralysis.⁶ Utilizing this model, we attempted to follow the formation and rupture of intracranial aneurysms using a 3-T MRI system.

Materials and Methods

All of the animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Hamamatsu University School of Medicine, Hamamatsu, Japan. We followed Guidelines for Proper Conduct of Animal Experiments issued by Science Council of Japan. The experiments were performed in accordance with the ARRIVE guidelines.

The Mouse Model of Intracranial Aneurysm and Subarachnoid Hemorrhage

Intracranial aneurysms were induced in 7-week-old male mice (C57BL/6J; Japan SLC, Hamamatsu, Japan) using a previously described method.^{2, 6, 7, 8} We combined systemic hypertension with a single injection of elastase into the cerebrospinal fluid in the right basal cistern.

To induce systemic hypertension, we used deoxycorticosterone acetate (DOCA) salt hypertension as previously described.⁹ The mice underwent a unilateral nephrectomy. One week later, a DOCA pellet (2.4 mg/day, Innovative Research of America, Sarasota, FL, USA) was implanted in the subcutaneous tissue through the dorsal midline incision. 1.0% sodium chloride drinking water was initiated on the same day as the DOCA pellet implantation.^{7, 9} The mice also received a single injection of elastase solution (35 milli-units Elastase: E7885, Sigma Aldrich dissolved in 2.5 μL of phosphate-buffered saline) into the cerebrospinal fluid in the right basal cistern on the same day as the DOCA pellet implantation.^{6, 7, 8} The nephrectomy was performed under general anesthesia with 2% isoflurane. The elastase injection was performed under general anesthesia induced by an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg).

The mice were housed individually after the induction of the aneurysms to avoid wound infection and dehiscence. The mice were maintained at standard housing conditions (temperature, 24±2 °C; relative humidity, 60±10% light/dark cycle, 0700 to 1900 hours, 12 hours) with water and food ad libitum.

A daily neurologic examination was performed using a previously described method.^{10, 11, 12} Neurologic symptoms were scored as follows: 0, normal function; 1, reduced eating or drinking activity demonstrated by a body weight loss of 2 g (approximately 10%) over 24 hours; 2, flexion of the torso and forelimbs on lifting the animal by the tail; 3, circling to one side with a normal posture at rest; 4, leaning to one side at rest; and 5, no spontaneous activity. The mice were euthanized when they developed neurologic symptoms (score 1 to 5). All asymptomatic mice were euthanized 14 days after aneurysm induction.

Brain Inspection After Euthanasia

After euthanasia, the mice were perfused with ice-cold saline followed by perfusion with a bromophenol blue dye solution (2 mg/mL) dissolved in phosphate-buffered saline and gelatin mixture (20%) at physiologic pressure (approximately 80 mm Hg). The bromophenol blue dye and gelatin mixture was used to visualize major cerebral arteries. Brain samples were assessed for the presence of intracranial aneurysm formation and subarachnoid hemorrhage under a dissecting microscope ( × 10). Aneurysms were operationally defined as a localized outward bulging of the vascular wall with a diameter greater than that of the parent artery as previously described.^{2, 7}

We used non-enhanced magnetic resonance angiography (MRA) for the intracranial aneurysm observation and T2-weighted imaging for subarachnoid hemorrhage detection. To verify the quality of the images, we used masked observer examinations.

The Magnetic Resonance Imager and Animals

We used a 3-T magnetic resonance imager (Signa HDxt; GE Healthcare, Milwaukee, WI, USA) at the animal experimentation facility at the Hamamatsu University School of Medicine. The gradient specifications of the MR system were as follows: amplitude for each axis: 50 mT/m; slew rate for each axis: 150T/m/s. For the mouse cerebral artery imaging, a saddle radiofrequency coil with a 40-mm inner diameter (Takashima Seisakusho, Tokyo, Japan) was used for the radiofrequency reception. General anesthesia was induced with 3% isoflurane in a 2 : 1 air/oxygen mixture and was maintained with 0.8 to 1% isoflurane.

The mouse was immobilized in an acrylic cradle inside the coil. A silicone tube with an anesthesia mask was connected to an anesthesia machine outside the MRI scanning room. The anesthetic agent was delivered via tubing. The adequate respiratory condition was visually confirmed under the steady state of anesthesia with isoflurane (0.8 to 1%) immediately before the imaging session. Inside the coil, a heat gel (5.5 × 7 × 0.7 cm) was used to cover the back side of the mouse as shown in the Online Supplementary Data. Our preliminary study that was conducted in the MRI machine without actual imaging showed that the average temperature of the heat gel was approximately 40.0 °C and that the body temperature of the mice remained above 35.0 °C over the 15-minute period.

Magnetic Resonance Angiography for Intracranial Aneurysm Observation

MRA was performed the day before the elastase injection and on days 1, 3, 7, 10, and 14 after the elastase injection. In selected mice, additional images were obtained when the mice became symptomatic. All the MRA images and data were stored on a server for the subsequent masked observer examinations.

The initial localizers were obtained in three orthogonal planes using a gradient echo sequence. After the initial localizers were acquired, time-of-flight angiograms were obtained using a three-dimensional (3D) spoiled gradient echo sequence with the following imaging parameters: repetition time (TR), 25 milliseconds; echo time (TE), 4.8 milliseconds; acquisition matrix size (slice × phase × frequency), 113 × 288 × 192; field of view, 6 × 8 cm; slice thickness, 0.2 mm; slice gap, 0.1 mm; flip angle, 20° bandwidth, 15.63 kHz; and number of averages, one. The resolution of the MR angiography was calculated as 0.28 mm with an acquisition matrix of 288 × 192. The MRA was sampled in a slab with an acquisition time of 3.7 minutes. All of the data were zero-filled to 512 × 512 for reconstruction. In addition to the standard MRA used for serial imaging and masked observer testing, we performed additional MRA for 3D reconstruction using the following setting: acquisition matrix size (slice × phase × frequency), 113 × 512 × 256; bandwidth, 31.25 kHz; and number of averages, three. The resolution of the MRA was calculated as 0.16 mm with an acquisition matrix of 512 × 256. The MRA images for the 3D reconstruction were sampled in a slab with the total acquisition time of 14.3 minutes.

T2-Weighted Imaging for Subarachnoid Hemorrhage Detection

Subarachnoid hemorrhage imaging was performed subsequently to the MRA. Similarly to MRI, the images were stored for the subsequent masked observer examinations.

To visualize the hemorrhagic changes in the brain tissue, T2-weighted images were acquired using a fast spin echo sequence in coronal and sagittal sections with the following imaging parameters: TR, 3,500 ms; TE, 85 ms; acquisition matrix size, 256 × 224; field of view, 4 × 4 cm; slice thickness, 1.2 mm; slice gap, 0.2 mm; echo train length, 12; bandwidth, 10.42 kHz; and number of averages, four. The number of slices was set at 11.

Masked Observer Examination

A masked observer examination was conducted to verify the quality of the images. The two masked observers (Tetsuro Kimura and YK) were trained using serial image sets of representative intracranial ruptured and unruptured aneurysms and brain vessels without intracranial aneurysms. The observers were trained by an investigator who was familiar with the mouse model of intracranial aneurysms (HM). After training, the observers assessed the stored MRI data. The analysis of the MR angiogram data was conducted on the day after the MRA imaging session. The assessment was made on the maximum intensity projection angiogram and the single slice vessel outlines of the multi-slice data set. The observers' results were compared with histologic diagnoses obtained after euthanasia. To assess the presence of subarachnoid hemorrhage, the observers assessed the stored T2-weighted images. The observers' results were compared with neurologic symptoms observed in the mice with subarachnoid hemorrhage and postmortem brain inspection result.

Results

Ten mice underwent the aneurysm induction. Subsequently, five mice (50%) developed neurologic symptoms associated with aneurysmal rupture (score 1 to 5). When the brains of these mice were inspected, all exhibited intracranial aneurysms with subarachnoid hemorrhage. Fourteen days after the aneurysm induction, the remaining asymptomatic mice (5/10) were euthanized. Among the asymptomatic mice, one had an unruptured aneurysm and four had no aneurysms. None had subarachnoid hemorrhage. Therefore, the incidence of ruptured aneurysms was 50%, and the incidence of unruptured aneurysms was 10%, consistent with previous reports using this model.^{2, 6, 7, 8}

Intracranial Aneurysm Observation Using Magnetic Resonance Angiography

We conducted a total of 54 MRA imaging sessions using the 10 mice. The MRA scanning time for each mouse was 3.7±0.5 minutes. There were no MRA-associated mortalities.

In all of the mice with aneurysms (6/10), we were able to detect aneurysm formation using serial MRA imaging. Aneurysm formation was detected on average 4.5 days after aneurysm induction.

Figure 1 shows representative serial MRA images. To depict the entire Circle of Willis and its major branches in one image, we constructed a maximum intensity projection image from 113 slices at 100-μm intervals for each mouse. Mouse #1 and #7 had ruptured aneurysms; mouse #8 had no aneurysms.

Figure 1.

Representative serial magnetic resonance images of a mouse with an unruptured aneurysm. In the angiogram, the deformity of the cerebral arteries was evident after the elastase injection. An aneurysm was clearly seen at the distal portion of the right middle cerebral artery, located inside the white circle. An enlarged aneurysm is observed on day 12. A mature aneurysm is observed at the same location in the brain sample (lower panels).

In mouse #1 (Figure 1), we detected a deformity of the Circle of Willis after the aneurysm induction. Abnormal flexions of the brain vessels were observed. An aneurysm was detected at the distal portion of the right middle cerebral artery 7 days after the aneurysm induction. The aneurysm size appeared to be constant from days 7 to 10. On day 12, the mouse showed neurologic symptoms. Immediately after the final MRA session, the mouse was euthanized. When we harvested and inspected the brain tissue, we found an aneurysm at the distal portion of the right middle cerebral artery (Figure 1, lower panels). This location of the aneurysm was the same as that detected by the serial MRA.

Figure 2 shows a 3D reconstruction of the cerebral arteries of a mouse that developed an intracranial aneurysm. Both the maximum intensity projection and 3D reconstructed images clearly showed aneurysm formation at the same location as that detected by the autopsy.

Figure 2.

Two- and three-dimensional reconstruction of an aneurysm. Representative images of an intracranial aneurysm (left) and a maximum intensity projection image of magnetic resonance angiography (MRA, middle) and a three-dimensional (3D) reconstructed image (right). A 3D reconstructed movie is available online supplement. For the 3D reconstruction, longer acquisition time (14.3 minutes) was applied compared with the regular MRA session used for serial imaging and masked observer testing (3.7 minutes).

In mouse #8 (Supplementary Figure S2), no changes were observed in the cerebral arteries from the day before injection until 14 days after aneurysm induction. The Circle of Willis was bilaterally symmetric, and no abnormal curvatures of the brain vessels were observed. When this mouse was euthanized on day 14 and the cerebral arteries were inspected, no abnormal changes were evident.

Table 1 summarizes the findings of the MRA and the direct observation of the brain after euthanasia. Using the MRA, two masked observers were able to detect aneurysm formation in all the six mice that showed aneurysms under direct observation after euthanasia. However, the two observers diagnosed aneurysm formation in three mice that were later confirmed not to have aneurysms by direct inspection. The combined sensitivity of the serial MRA to detect intracranial aneurysms was 100% the specificity was 62.5%. The rupture status did not affect the sensitivity or specificity.

Table 1. Detection of aneurysm formation and aneurysmal subarachnoid hemorrhage.

ID	Aneurysm formation			Aneurysmal rupture
	Aneurysm detection by MRA		Confirmation after autopsy	Rupture detection by T2 MRI		Neurologic score	Confirmation after autopsy
	Observer 1 (day; location)	Observer 2 (day; location)		Observer 1	Observer 2	(score; day)	Rupture: yes or no; day
1	7; Right MCA	7; Right MCA	Right MCA	Day 10	Day 10	1; 12	Yes; 12
2	No aneurysm	10; Left PCA	No aneurysm	No rupture	Day 10	0	No; 14
3	10; Right MCA	No aneurysm	No aneurysm	No rupture	No rupture	0	No; 14
4	3; Right ACA	3; Right ACA	Right ACA	Day 10	Day 10	4; 10	Yes; 10
5	3; Right MCA	3; Right MCA	Right MCA	Day 7	Day 7	1; 7	Yes: 7
6	1; Right MCA	1; Right MCA	Right MCA	Day 5	Day 5	1; 5	Yes; 5
7	3; Right ACA	3; Right ACA	Right ACA	Day 7	Day 7	1; 10	Yes; 10
8	No aneurysm	10; Right MCA	No aneurysm	No rupture	Day 14	0	No; 14
9	No aneurysm	No aneurysm	No aneurysm	No rupture	No rupture	0	No; 14
10	10; Bilateral PCA	10; Bilateral PCA	Bilateral PCA	No rupture	No rupture	0	No; 14

Abbreviations: ACA, anterior cerebral artery; MCA, middle cerebral artery; MRA, magnetic resonance angiography; MRI, magnetic resonance imaging; PCA, posterior cerebral artery.

Neurologic symptom score:

0: Normal function.

1: Reduced eating or drinking activity demonstrated by a body weight loss of 2 g over 24 hours.

2: Flexion of the torso and forelimbs on lifting the animal by the tail.

3: Circling to one side with a normal posture at rest.

4: Leaning to one side at rest.

5: No spontaneous activity.

Subarachnoid Hemorrhage Detection Using T2-Weighted Imaging

To detect subarachnoid hemorrhage, we performed 54 T2-weighted imaging sessions in addition to the serial MRA. The imaging time for each mouse was 4.8±0 minutes, and there were no T2-weighted imaging-associated mortalities.

Mouse #7 (Figure 3) showed a small low-intensity area in the middle portion of the brain on day 7. However, no neurologic symptoms were observed on that day. On day 10, the low-intensity area became larger, and the mouse developed neurologic symptoms. The mouse was euthanized immediately after the imaging session. A direct inspection of the brain tissues revealed an apparent subarachnoid hemorrhage at the same location as the low-intensity area.

Figure 3.

Representative serial magnetic resonance images of a mouse with aneurysmal subarachnoid hemorrhage. In the T2-weighted images on day 7, a small low-intensity area is observed in the middle portion of the brain (white circle), but no neurologic symptoms were observed on the same day. By day 10, the low-intensity area was enlarged (white circle).

The asymptomatic mice did not exhibit any abnormalities in the T2-weighted images or upon direct visualization after euthanasia.

Table 1 summarizes the findings of T2-weighted MRI and direct observation of the brain after euthanasia. All five symptomatic mice presented a low-intensity area on T2-weighted imaging. However, in two mice, the low-intensity area was detected before the mice became symptomatic. In these mice, the growth of the low-intensity area coincided with the onset of symptoms. The location of the low-intensity area matched that of the aneurysmal subarachnoid hemorrhage revealed by the inspection of the brain after euthanasia. The combined sensitivity and specificity of the masked observer test were 100% and 80%, respectively.

Discussion

In this study, we demonstrated the feasibility of using a conventional 3-T MRI system with a saddle coil but no contrast agent to detect changes in cerebral arteries and subarachnoid hemorrhage in mice. We were able to longitudinally follow the development of intracranial aneurysms and aneurysmal subarachnoid hemorrhage in mice without causing significant morbidity or mortality. This technique has the potential to simplify serial imaging of mouse cerebral arteries and make this previously costly method more accessible.

This technique has several notable attributes. First, it does not require any special modifications of the conventional clinical 3-T MRI system. The only special devices necessary are a saddle coil and a standard small animal anesthesia system. Second, the image acquisition time is sufficiently short to accommodate a relatively large sample size for preclinical studies. The average image acquisition times for MRA and T2-weighted imaging were 3.7±0.5 minutes and 4.8±0 minutes, respectively. This short image acquisition time is suitable for animals of which general condition is compromised because of physiologic or pathologic perturbations induced to generate various disease models. Fourth, at least in this series, there were no imaging-associated mortalities. This is partly because this technique does not require invasive vascular cannulation or a potentially toxic contrast agent. Finally, the imaging quality was sufficient to detect the changes in cerebral arteries that occur during aneurysm formation.

MRA without a contrast agent was sufficient to detect the remodeling of cerebral arteries in mice. Even before aneurysm formation was detected, significant changes in the cerebral arteries were evident. We observed an increased tortuosity of the cerebral artery before aneurysm formation. A similar change was reported in a model of cerebral aneurysm in rabbits.¹³ The MRA technique tested in this study appears to be sufficiently accurate and reproducible for future preclinical studies in mice. This technique can be easily applied to other species, including rats. Its simplicity and low cost make this technique readily accessible to many researchers, particularly for studies requiring serial imaging of the cerebrovasculature in a relatively large number of mice.

The noise in the MR angiogram maximum intensity projection can be reduced by adjusting the flip angle. Theoretically, a larger flip angle may reduce the noise. However, a larger flip angle can cause saturation effects, thereby reducing the signal intensity of the distal portion of the cerebral arteries. Empirically, we found the flip angle of 20 degrees that was used in our main study to be a compromise for achieving the overall imaging quality while reducing the noise. Although it was an advantage of our protocol that contrast agents were not required for the visualization of the cerebral arteries, a contrast agent may be useful for detecting early changes in the blood–brain barrier after subarachnoid hemorrhage.

It should be noted that we used T2-weighted imaging instead of T2-fluid attenuated inversion recovery imaging to detect changes associated with aneurysmal subarachnoid hemorrhage. In clinical settings, the T2-fluid attenuated inversion recovery imaging facilitates the excellent detection of superficial subarachnoid hemorrhage, whereas it demonstrates a high detection rate for centrally located hemorrhage, i.e., intraventricular and interhemispheric.¹⁴ In our preliminary study (data not shown), we tested the ability of T2-fluid attenuated inversion recovery and susceptibility-weighted imaging to detect subarachnoid hemorrhage in mice without success. This may be because the mouse subarachnoid volume is animal size-dependently small.¹⁵ In addition, the auditory meatus in mice is disproportionally large compared with that in humans. A large auditory meatus interferes with the T2-fluid attenuated inversion recovery and susceptibility-weighted imaging. Conversely, T2-weighted imaging yielded images of a quality sufficient for detecting aneurysmal subarachnoid hemorrhage. When MRA and T2-weighted imaging are combined, the entire course of the development, growth, and rupture of intracranial aneurysms can be longitudinally followed in mice.

This study has a number of limitations. First, we were able to visualize the relatively large pathologic remodeling of cerebral arteries, such as intracranial aneurysms. However, considerably smaller intracranial aneurysms and pathologic changes in the cerebral arteries may be difficult to distinguish. Extending the acquisition time of the MRA yields images of a much higher resolution; however, extending the duration of imaging and anesthesia may be harmful for sick mice and may increase imaging-associated mortality. To visualize considerably smaller pathologic remodeling of cerebral arteries in a short acquisition time, further optimization of MRA settings may be required. Second, in the masked observers study, the observers diagnosed the expansion and shape change in the cerebral arteries as intracranial aneurysms. This type of error may reduce the method's specificity for detecting intracranial aneurysm.

The aneurysm formations in this mouse model showed significant variations in their locations and sizes.² Although such variation reflects actual clinical scenarios, they increase the difficulty of the MRI-based detection of aneurysms. Our results showed that the current setup of our MRI imaging was sufficient for detecting aneurysms at variable locations. However, because of the image distortion caused by the respiratory cycle and cardiac oscillation, the size measurement using the current MRI setting may not be accurate enough to use size as one of the key outcomes.

In summary, we showed the feasibility of using a conventional 3-T MRI system for serial MRA without a contrast agent combined with T2-weighted imaging to follow anatomic changes in the cerebral arteries. Using this technique, we were able to detect the formation and rupture of intracranial aneurysms in mice. We were able to identify aneurysms before they ruptured. These protocols can be used for the stratification of animals based on the imaging findings: thus, the experimental treatments can be targeted to specific stages of disease or specific attributes of aneurysms. This technique has applications in future cerebrovascular research involving the longitudinal follow-up of cerebral arteries.

The authors declare no conflict of interest.