Longitudinal investigation of ischemic stroke using magnetic resonance imaging: Animal model

Abstract

BACKGROUND:

Arterial embolism is a major cause of ischemic stroke. Currently, digital subtraction angiography (DSA) is the gold standard in clinical arterial embolization examinations. However, it is invasive and risky.

OBJECTIVE:

This study aims to longitudinally assess the progression of carotid artery embolism in middle cerebral artery occlusion animal model (MCAO) using magnetic resonance imaging (MRI) techniques.

METHODS:

Turbo spin echo (TSE), time of flight magnetic resonance angiography (TOF-MRA) and diffusion weighted magnetic resonance imaging (DWI) were used to evaluate the image characteristics of cerebral tissues at 1, 2, 3, 7, 14, 21 and 28 days after MCAO microsurgery on Sprague-Dawley (SD) rats. Quantitative analysis was performed and compared in MCAO hemisphere and contralateral normal hemisphere. Furthermore, pathologic section using triphenyl tetrazolium chloride (TTC) stain was performed as well.

RESULTS:

TOF-MRA showed carotid signal void in the embolism side, which is evidence of artery occlusion. The used MRI techniques showed that edema gradually dissipated within one week, but there was no significant change afterwards. The time-varying signal intensity of MRI techniques in MCAO hemisphere changed significantly, but there were no significant changes in contralateral normal hemisphere. Cerebral injury was also confirmed by analysis of pathology images.

CONCLUSIONS:

The MCAO animal model was successfully established on SD rats using the microsurgery to assess arterial embolization of intracranial tissue injury.

Keywords

Time of flight magnetic resonance angiography diffusion weighted magnetic resonance imaging apparent diffusion coefficient Sprague-Dawley rat middle cerebral artery occlusion animal model ischemic stroke

1 Introduction

Stroke is one of the most common causes of human disability and death globally. Artery embolism is a major cause of ischemic stroke and may result in ischemic heart disease such as myocardial infarction as well. Even though the brain’s blood supply is just temporarily interrupted for a few minutes causing cerebral cell death, permanent neurological deficits might be induced due to arterial embolism. In total, 6.3 million people died of stroke which is one of the most common causes of death after coronary artery disease, and about 3.0 million deaths resulted from ischemic stroke in 2015 [1]. Furthermore, in one study, about half of the patients who had a stroke passed on within one year [2]. At present, digital subtraction angiography (DSA) is still the gold standard in routine clinical arterial-venous disease [3, 4]. However, DSA is an invasive and high-risk technique. In comparison with other imaging modalities, such as DSA, computed tomography (CT) etc., magnetic resonance imaging (MRI) is suitable to perform longitudinal studies due to no harmful x-ray and contrast agent prone to allergic effect used, and has superior tissue contrast and diagnostic accuracy as well. As far as we know, there is no article regarding longitudinal investigation of ischemic stroke using MRI technique. It is still a challenging issue that needs to be solved in how to develop an effective treatment approach. The experimental stroke model of focal cerebral ischemia in rats was introduced to meet the study needs. Middle cerebral artery occlusion (MCAO) models were frequently used to mimic cerebral ischemia, which were classified into permanent or transient occlusion methods [5, 6]. In 1986, Koizumi first proposed an animal MCAO model to induce ischemic stroke using endovascular filament, namely intraluminal suture MCAO [6].

3T high-field whole-body magnetic resonance scanners have been approved for clinical diagnosis by the FDA. A higher magnetic field can provide higher signal-to-noise ratio (SNR), thus allowing magnetic resonance imaging with high spatial resolution, and reduce scan time. 3T magnetic resonance scanner has been shown to be an effective method to assess brain function, detect brain lesions and cardiac structure, and so on. The diagnostic accuracy can be increased by Gd-base contrast-enhanced MR Imaging technique [7–9]. A middle cerebral artery occlusion animal model in rats was proposed to investigate brain tissue progression and image signal variations after carotid arterial embolization surgery using turbo spin echo (TSE) [10], time of flight magnetic resonance angiography (TOF MRA) [11–14], and diffusion-weighted magnetic resonance imaging (DWI) [10] techniques. Furthermore, the image characteristics and correlations between these two techniques in an SD rat MCAO model were compared as well. Nowadays, cerebrovascular disease has become one of the major causes of death, which is an important issue in clinical settings. MRI and MRA are capable of more timely detection of cerebral vascular diseases, and are now more widely used in medical research and clinical diagnosis. Currently, MRI and MRA techniques are often used to detect asymptomatic lacunar infarcts, un-ruptured aneurysm and peripheral arterial occlusion disease (PAOD).

Although DSA is commonly used for clinical examination of aneurysms, it is invasive and risky when performed via femoral artery surgery [15–17] or via ear artery or via vein [18, 19]. A 3-D volumetry was proposed to measure the aneurysm size in order for endovascular microsurgery planning and follow-up in clinics to be accurately performed. Several research teams have proposed the use of 1.5 T MRA to replace the traditional DSA [20, 21]. 3 T MRA could provide higher resolution and signal-to-noise ratio (SNR) image than 1.5 T, which results in higher accuracy in assessing aneurysms [22]. George et al compared 3D MRA and 2D-DSA in an experimental rabbit model at 3 T and found the superior correlation between 3D MRA and 2D-DSA for assessing size of aneurysms and surrounding vessels [23]. Hence, 3D TOF MRA was adopted in this study to demonstrate whether MCAO surgery was successful.

DWI technique is based on diffusion properties of water molecules between tissues [24], as proposed in 1965 [25]. Regional tissue contains more intensive cells, so water molecular movement will be restricted more. If regional tissue contains more sparse cells or cell necrosis, water molecular movement will be restricted less [26–28].

The signal intensity of DWI can be formulated as $S = S_{0} \times e^{- γ^{2} G^{2} δ^{2} (Δ - \frac{δ}{3}) \cdot D} = S_{0} \times e^{- b \cdot D}$ (1)

Where γ is the gyromagnetic ratio, δ is the duration of applied diffusion gradient magnetic field, Δ is the time interval between two diffusion gradients, G is the strength of diffusion gradient, D is the apparent diffusion coefficient (ADC) and b-value is diffusion gradient parameter in unit of s/mm ² which is defined as [17] $b = γ^{2} G^{2} δ^{2} (Δ - \frac{δ}{3})$ (2)

The higher the b-value, the more sensitive the water molecule diffusion. There is higher signal decay for tissue with lager b-value.

ADC value can be obtained by scanning with two different b-values, namely b ₁ and b ₂, $D = - \frac{1}{b_{1} - b_{2}} \times (\ln \frac{S (b_{1})}{S (b_{2})})$ (3) where S(b ₁)is the signal intensity of the first scan with b ₁ and S(b ₂) is the signal intensity of the second scan with b ₂. ADC mapping denotes ADC value for each pixel.

The purpose of this study was to evaluate the intracranial tissues damage and recovery processes of an MCAO Sprague-Dawley (SD) rat model and derive correlations of tissue characteristics between controls and an MCAO group using MRI techniques. The cerebral arterial thrombosis MCAO animal model was induced using microsurgery in SD rats. TOF MRA, TSE and DWI of the MCAO SD rats after microsurgery were obtained to compare imaging characteristics, stroke formation and progression. It can be applied to medical research modalities including brain ischemia, stroke, and pathology on the MCAO animal model.

2 Materials and methods

Longitudinal assessments of the imaging features for MCAO SD rats using T2-weighted TSE, 3D TOF, and diffusion-weighted MRI were performed on a weekly basis for a period of one month. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC-A102-03), Kaohsiung Armed Forces General Hospital.

2.1 MCAO SD rat model

Twenty-one 6-week-old normal male SD rats weighing 150 g to 200 g were used as the experimental animal in this study, which were performed a middle cerebral arterial occlusion microsurgery to induce ischemia in the right cerebral cortex and mimic cerebral ischemia stroke in humans. In order to prevent the reperfusion and further injury to the cerebral tissue, permanent MCAO method was adopted. The cerebral ischemic hemisphere was referred to as the MCAO group, while the contralateral side was referred to as the control group. Gas anesthesia machine with Isoflurane (Abbott Laboratories, North Chicago, IL) was used for microsurgery.

Microsurgery was performed by inserting paraffin-coated nylon wire to induce ischemia in the right cerebral cortex in a sterile controlled environment at room temperature 23±0.2°C. The detailed procedure of MCAO microsurgery was as follows:

1.
The SD rats were anesthetized with 1:1 Isoflurane (Halocarbon Products Corporation, NJ, USA) and oxygen (99.5%) mixture in a homemade anesthesia box.
2.
The rat was taken out from the anesthesia box after the rat was fully unconscious, then the surrounding fur around the neck of SD rat was shaved off.
3.
The head of the rat was masked for anesthesia, positioned in supine orientation, and the four limbs secured with 3Mtrademark Microporetrademark surgical tape and rubber bands.
4.
After the surgical site was sterilized with 75% ethyl alcohol (Kespol, Kaohsiung, Taiwan), a surgical scissor was used to make a 1 cm midline neck incision.
5.
Forceps were used to push aside the upper fascia and muscles of the neck until the carotid artery was visible, then the common carotid artery (CCA) and vagal nerves were separated with forceps and the platysma proximal to the internal carotid artery (ICA) and the external carotid artery (ECA) was pushed aside.
6.
The common carotid artery and the external carotid artery were ligatured by polypropylene suture.
7.
A 25 mm long paraffin-coated nylon wire was inserted from the ECA trace to ICA into the brain causing obstruction.
8.
After ICA was ligatured using polypropylene suture, the muscle outer skin was sutured.
9.
After embolization surgery, disinfection with 0.125 cc/kg antibiotic “Cefazolin injections” (1 gm, Yungshin Pharm. Ind. Co. Ltd., Taiwan) was injected at the surgical site daily for a week.

2.2 MRI protocol

A Zoletil 50 (20 mg/kg)/Rompun (5 mg/kg) mixture via muscle injection was used for MRI scanning experiments. Siemens Magnetom Skyra 3T whole body MR scanner (Siemens Medical Solutions) equipped with16-Channel high resolution knee coil was used for regular scans. Daily MR scanning was performed for the first three days and weekly for a period of 4 weeks after the micro-surgery longitudinally. The pulse sequences included T2 TSE, TOFMRA and DWI scans. The scanning parameters of TSE T2W, 3D TOF, and DWI-EPI pulse sequences were optimized for SD rats as listed in Table 1. The MR scanning procedures are summarized as follows:

Table 1
MRI scanning parameters used in routine experiments of MCAO SD rat

Parameters	3D TOF	TSE T2	DWI-EPI
TE(ms)	8.75	88	80
TR(ms)	29	2500	8000
Flip angle (°)	18°	90°	30°
Bandwith (kHz)	65	149	760
Matrix	512×512	192×192	124×124
NEX	2	7	4
Phase FOV	1	1	1
FOV(mm²)	94×94	94×94	94×94
Slice Thickness	0.5	2	2
b-value	–	–	0/1000
diffusion directions	–	–	3

A 3-plane localizer scan. (to acquire multi-slice axial, coronal and sagittal images for localization)

Calibration Scan.

T2-weighted (T2W) Turbo Spin Echo (TSE).

3D Time of Flight (TOF) angiography.

Diffusion Weighted Imaging - Echo Planar Imaging (DWI-EPI).

2.3 Image processing and statistical analysis

All acquired 3D TOF image data were transferred to the Sygno imaging workstation (Siemens) to perform MRA imaging reconstruction. In addition, ImageJ software was used for ROI measurements on T2 TSE, DWI images and ADC maps. Statistical analysis was performed using SPSS statistics software 24.0, with significance level set at 0.05. The quantitative analysis, correlation assessment and comparison of brain development between the MCAO hemisphere and contralateral control hemisphere of MCAO SD rats were measured. Cerebral tissue injury, recovery and change over time was evaluated and compared individually between the control group and MCAO group of SD rats after microsurgery. The differences between these two groups were analyzed as well.

2.4 Autopsy histopathology analysis

Triphenyl tetrazolium chloride (TTC) stain was adopted to perform SD rat cerebral autopsy histopathology to investigate the progression, and interpret structural variations of infarction and cerebral ischemia. After anesthesia, SD rat brains were dissected and removed, and 2 mm coronal slices were cut within a short time (<10 minutes). Afterwards, these slices were soaked in 2% fresh TTC/normal saline solution and put inside a 37°c-heated oven for 15 minutes. TTC reacts with dehydrogenase in the survival cells of normal tissues and turns red, while TTC can’t react with ischemic tissue because of the decreases of dehydrogenase activity and appears as a paler color. After the completion of TTC stain, the pathologic sections were photographed using a digital camera.

3 Results

Figure 1 shows activity of normal SD rats and MCAO SD rats. When a normal SD rat turned the head around, SD rats could retain straightness (top); however, MCAO SD rats could only crook the head to the left (below). It implies that stroke was formed on the right brain. Figure 2 shows the march-forward track of MCAO SD rats. March-forward track of normal SD rats was somewhat in a straight line, and that of MCAO SD rats showed precession counterclockwise. It implies that stroke was formed on the right brain as well.

Fig.1

Activity of normal (top) and MCAO SD rats (below).

Fig.2

March trajectory of the MCAO SD rat.

MRA images of normal control (1st row) and MCAO SD rat (2nd row) left: AP view; middle: PA view; right: lateral view are shown in Fig. 3. It shows signal void in the right carotid arteries, which is evidence of successful artery occlusion. The hyper-signal region volume at different time frames after MCAO microsurgery is shown in Fig. 4, where 4-week survival of MCAO rats (ν), 1-week survival of MCAO rats (ν), and 3-day survival of MCAO rats (ν) are shown.

Fig.3

MRA images of normal (1st row) and MCAO SD rat (2nd row) left: Top view; middle: bottom view; right: lateral view.

Fig.4

Hyper-signal region volumes at different time frames after MCAO microsurgery. Color bar denotes 4-week survival MCAO rats (ν), 1-week survival MCAO rats (ν) and 3-day survival MCAO rats (ν).

T2-weighted TSE series images of MCAO rat brain at different time frames are shown in Fig. 5. It shows that the hyper-intensity region becomes smaller in the first 3 days after microsurgery, and does not change significantly after the first week.

Fig.5

T2 TSE series images of the MCAO rat brain at different time frames.

Figure 6 shows the time-varying signal intensity curves of T2 TSE at different time frames for control (131∼143) and MCAO hemispheres (198∼271). The time-varying signal intensity curves of control hemispheres showed no significant differences over time; however, the average hyper-intensity of stroke regions continuously increased over time after one week for MCAO hemispheres, which had significant differences over time with significant differences between control and MCAO hemispheres.

Fig.6

Mean signal-intensity curves of normal and MCAO hemispheres of the SD rat brain obtained from T2 TSE series images at different time frames.

Diffusion-weighted series images of MCAO rat brain at different time frames are shown in Fig. 7. It also shows that the hyper-intensity region becomes smaller in the first 3 days after microsurgery, and does not change significantly after the first week. Figure 8 shows the time-varying signal intensity curves of DWI-EPI at different time frames for control hemispheres (97∼104) and MCAO hemispheres (96∼231). The time-varying signal intensity curves of control hemispheres showed no significant differences over time; however, those of stroke regions in MCAO hemispheres had significant differences over time with significant differences between control and MCAO hemispheres.

Fig.7

DWI series images of the MCAO rat brain at different time frames.

Fig.8

Mean signal-intensity curves of normal and MCAO hemispheres of the SD rat brain obtained from DWI series images at different time frames.

ADC series maps of MCAO rat brain at different time frames are shown in Fig. 9. It shows an evidence of edema because the hypo-ADC regions were smaller than those of normal ADC regions in the first 3 days after microsurgery. The ADC of stroke regions increased rapidly, and then gradually increased after the first week. On the contrary, no significant change of ADC over time on control hemispheres is shown. The time-varying ADC curves of MCAO rat brain at different time frames for control hemispheres (665×10^–6∼731×10^–6 s/mm ²) and MCAO hemispheres (397×10^–6∼1608×10^–6 s/mm ²) are shown in Fig. 10. The time-varying ADC values of control hemispheres showed no significant differences over time; however, those of stroke regions in MCAO hemispheres had significant differences over time with significant differences between control and MCAO hemispheres.

Fig.9

Series ADC maps of the MCAO rat brain at different time frames.

Fig.10

ADC curves of normal control and MCAO hemispheres of the SD rat brain obtained from series ADC maps at different time frames.

Figure 11 shows the TTC staining of SD rat brain sections, survival cells converts TTC to red, while injured tissue area corresponding to infarction or cerebral ischemia becomes a paler color.

Fig.11

TTC stain of SD rat brain section. Left: normal brain; right: MCAO brain.

4 Discussion

This study has successfully established a cerebral arterial thrombosis SD rat animal model using microsurgery to assess intracranial tissue injury and recovery process after arterial embolization microsurgery. The MR image characteristics of SD rat cerebral tissues after arterial embolization has been explored using a number of MRI techniques including T2 TSE, TOF MRA, DWI and ADC mapping in MCAO rats. The activity of normal and MCAO SD rats (Fig. 1) and signal void in MRA reconstructed images (Fig. 3) demonstrated successful stroke induction due to MCAO.

Twenty-one SD rats were used to perform MCAO microsurgery. One SD rat died at the second day, one SD rat died at the third day, five SD rats died at the eighth day, four SD rats die at the ninth day, and ten SD rats lived up to 28 days after MCAO microsurgery. SD rats were classified into three groups according to the survival time, Group I within 3 days (n = 2), Group II within 1 week (n = 9, 8-9 days) and Group III after more than 4 weeks (n = 10). Figure 4 shows the volume of hyper-signal region at different time frames after MCAO microsurgery. The hyper-signal region was larger at the first three days, significantly shrank at day 7, and remained constant afterwards until the 4th week. This might be because edema occurred immediately after MCAO microsurgery then became gradually absorbed by cerebral tissues. Hence, the hyper-signal region is due to the MCAO microsurgery and the survival time might be negatively correlated to the volume of the hyper-signal region; the larger the volume, the shorter the survival time. It was noted that the volume of the hyper-signal region tended to be smaller for SD rats with longer survival time. The volume of the hyper-signal region might reflect the severity of ischemic stroke. The induced edema was completely dissipated within 4 to 7 days. Evidence of the edema dissipation was shown between 3 to 7 days as found from the 4-week MCAO hemisphere.

Those who survived up to 4 weeks were used to perform image analysis, and the MCAO hemisphere and contralateral normal hemisphere were compared. Longitudinal magnetic resonance angiography, TSE and diffusion-weighted magnetic resonance imaging studies were scheduled on a regular basis after the micro-surgery daily for the first three days and then weekly for four weeks. The total experimental time lasted for a period of one month. Observation and comparison of MRI findings were carried out between the control hemisphere and MCAO hemisphere after thrombosis, including signal intensity changes over time, progression and recovery of injured brain.

T2 TSE and DWI techniques show the hyper-intensity region and hypo-intensity region in ADC maps became smaller over time within the first 3 days, and did not change significantly after the first week. The ADC curves of control hemisphere and ADC of MCAO hemisphere showed edema was discharged fully at third day after MCAO microsurgery.

The findings of behavior patterns, magnetic resonance imaging features and pathological tissue sections of MCAO SD rat are consistent. In conclusion, the MCAO SD rat model has been demonstrated to successfully induce a stroke. Stroke might not be effectively induced using the ligation SD rat model due to collateral circulation acting as a source to supply blood. The benefits of this research paper provide a free radiation and non-invasive method to observe the SD rat MCAO model longitudinally.

Another finding was the average signal intensity of injured thrombosis areas rapidly decreased in a week, and then gradually decreased after one week in DWI images. This might be due to the depolarization of cell membranes induced by ischemic-hypoxic cells after thrombosis, and extracellular water molecules drift into the cell resulting in edema. Then, edema quickly faded within a week resulting in DWI signal intensity decrease within a week in MCAO hemispheres as well, and the signal intensity is approaching that of the normal hemisphere a week later.

It shows that the ADC values of the stroke region increased rapidly in the first 3 days after microsurgery due to dissipation of edema in ADC maps. Liquefaction necrosis of cell results in the disintegration of cell membrane after a week, following by discharging water molecules inside the cell membrane, which enables a gradual increase of ADC value, but still higher than that of the normal hemisphere.

One of the limitations of this study was the low success rate (48%) of this study in keeping MCAO rats alive for 4 weeks.

5 Conclusion

This study has successfully established a cerebral arterial thrombosis SD rat animal model using microsurgery to assess intracranial tissue injury and recovery process after arterial embolization microsurgery. The SD rats’ behavior patterns, imaging features of MRI and pathological sections after MCAO microsurgery were consistent, which confirmed the MACO animal model on SD rats is an effective way to induce a stroke.

Conflict of interest

The authors declare that they have no conflict of interest. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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Longitudinal investigation of ischemic stroke using magnetic resonance imaging: Animal model

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1 Introduction

2.1 MCAO SD rat model

Table 1 MRI scanning parameters used in routine experiments of MCAO SD rat

2.4 Autopsy histopathology analysis

3 Results

5 Conclusion

Conflict of interest

References

Table 1
MRI scanning parameters used in routine experiments of MCAO SD rat