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Evaluation of Cerebral Blood Flow Changes in Focal Cerebral Ischemia Rats by Using Transcranial Doppler Ultrasonography
Ultrasound in Med. & Biol., Vol. 36, No. 4, pp. 595–603, 2010 Copyright Ó 2010 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/10/$–see front matter
doi:10.1016/j.ultrasmedbio.2010.01.005
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Original Contribution EVALUATION OF CEREBRAL BLOOD FLOW CHANGES IN FOCAL CEREBRAL ISCHEMIA RATS BY USING TRANSCRANIAL DOPPLER ULTRASONOGRAPHY LE LI, ZHENG KE, KAI YU TONG and MICHAEL YING Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong SAR, China (Received 12 May 2009; revised 31 December 2009; in final form 15 January 2010)
Abstract—Ischemic stroke is typically characterized by the disruption of cerebral blood flow. This study aimed to consecutively evaluate the cerebral blood flow changes in a focal ischemia rat model during the occlusionreperfusion procedure and along the recovery stage after stroke. In 12 Sprague Dawley (SD) rats, a middle cerebral artery occlusion/reperfusion (MCAo/r) surgery was conducted, which combines a permanent occlusion of the right common carotid artery (CCA), external carotid artery (ECA) and a transient occlusion of the right internal carotid artery (ICA) and middle cerebral artery (MCA) with a monofilament introduced from the proximal ICA towards the distal right ICA then removed after 90 min. Blood flow velocity (BFV) from the concerned arteries were measured using ultrasonography (13–4 MHz) at the basal stage before the surgery, after the reperfusion stage and during the post-stroke status. At reperfusion stage and after, BFV increased significantly in the left ICA and in the basilar artery (BA) (starting from post-24 h, p , 0.05 vs. basal). Moreover, BFV were reversed in the distal right ICA and reflow was recorded in the right MCA. Time-average maximum BFV in the right MCA at reperfusion and post-stroke 24–96 h was decreased significantly (p , 0.05 vs. basal). The reversed flow in the right ICA was enabled by the settlement of the collateral supply through the circle of Willis which consisted in higher BFV in the opposite ICA and in the BA still 24 h, although the proximal right ICA remain occluded. Ultrasound measurement of BFV helps to provide information on the redistribution of the blood flow supply after the onset of stroke. (E-mail: [email protected]) Ó 2010 World Federation for Ultrasound in Medicine & Biology. Key Words: Blood flow velocity, Focal cerebral ischemia, Rat, Ultrasonography.
perfusion, helping to describe the ischemic area and the surrounding penumbra. However, a consecutive examination of the cerebral hemodynamic during acute and subacute stroke is not possible with these techniques due to their insufficient temporal resolution and restricted access. Laser-Doppler flowmetry was used to monitor ipsilateral or local cerebral blood flow for occlusion and reperfusion in some animal studies (Ding et al. 2002; Sayeed et al. 2006). However, to increase the penetrability, sometimes the skull of the animal needed to be thinned that might cause unnecessary damage to the animal (Burnett et al. 2006). Transcranial Doppler (TCD) ultrasonographic assessment has been widely applied in the cerebral ischemia detection and evaluation. After Aaslid et al. (1982) introduced a 2 MHz Doppler system that allowed adequate penetration through the intact skull and demonstrated clinical useful recordings of blood flow velocities of the intracranial blood vessels, this noninvasive, reproducible and relatively inexpensive testing has become routine in the clinical management of the cerebral blood flow of patients with cerebrovascular disease or subarachnoid hemorrhage
INTRODUCTION Ischemic stroke happens when there is inadequate delivery of glucose and oxygen to the brain, which is caused by a critical reduction of cerebral blood flow (CBF), mostly due to the occlusion or stenosis of a brain-supplying vessel (Baron 1999; Choy et al. 2006). The reduction of cerebral flow results in energy failure and secondary biochemical disturbance, eliciting a robust inflammatory response in situ and this inflammatory process contributes to the development of cell injury in stroke (Dirnagl et al. 1999). Therefore, it is desirable to obtain information of blood flow changes from the actual cerebral perfusion deficit and on its time-course in patients suffering from stroke. Positron-emission tomography (PET) (Heiss and Podreka 1993) and magnetic resonance imaging (MRI) techniques (Besselmann et al. 2001; Yang et al. 2008) were used to evaluate cerebral diffusion and
Address correspondence to: Dr. Kai yu Tong, Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China. E-mail: [email protected] 595
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(Rigamonti et al. 2008). Some important and widespread applications of TCD have been reported in the detection of stenosis in the basal intracranial arteries (Demchuk et al. 2000); evaluation of extracranial internal carotid artery disease (Wilterdink et al. 1997), monitoring in cerebral embolism (Padayachee et al. 1986); and in the diagnosis of cerebral vasospasm (Proust et al. 1999), etc. However, animal studies are warranted if the mechanism and physiologic bases of ischemic stroke needed to be evaluated and these could help for a better understanding of the disease and better planning for neuroprotective treatment. The mainstream animal model of focal ischemic stroke is the middle cerebral artery occlusion/reperfusion (MCAo/r). In addition, the most conventional technique is the intraluminal model (Koizumi et al. 1999; Leung et al. 2006; Shimazu et al. 2004). However, this surgical procedure has a limitation in that the occlusion of the vessel could only be judged from experience and requires considerable skills. Although the markers on the filament could provide information on the depth of the insertion, the variation of individual animal anatomical structure could greatly affect the successful rate of the surgery. Modern ultrasound units and transducers with a smaller sample volume and an optimized focus allow measurements in small animal vessels with a high temporal resolution (Bonnin et al. 2008; Els et al. 1999). Therefore, two-dimensional (2-D) ultrasonography could help to illustrate the brain anatomy and make sure the filament is in the right position for improving the success rate of the model. The ultrasonographic measurement allows analysis of rapid changes in blood flow as early as arterial occlusion is performed and the evaluation could be repeated for a longitudinal survey at different time points. Ultrasound imaging on ischemic brain based on animal models has been reported on rabbit (Els et al. 1999) and rat pups (Bonnin et al. 2008). However, the detailed information of CBF velocity changes at different lesion time and the subacute stage (i.e., 16–24 h after surgery) as well as at the chronic stroke stage (160–168 h post-surgery, SoltanianZadeh et al. 2003) is not clear yet. In this current study, cerebral blood flow changes occurring during the whole procedure of cerebral ischemia-reperfusion and along the 4 days post-stroke recovery period from a focal ischemia rat model were evaluated by using highfrequency color Doppler ultrasonography, which may help to provide more information on the redistribution of blood flow supply after the onset of stroke. MATERIALS AND METHODS Twelve young male Sprague Dawley (SD) rats with body weight around 230–250 g were selected and housed individually during a 12 h light/dark cycle with access to
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food and water ad libitum in a temperature-controlled environment (20–22 C). Animals were anaesthetized with chloral hydrate (0.4 mg/kg, intraperitoneal) and all ultrasound assessments were conducted with an Esaote MyLab 70 X-view ultrasound unit in conjunction with a 13 to 4 MHz linear transducer (Esaote, Genova, Italy). The rat was placed on a stereotaxic frame and the probe was put on the skull of the rat after the fur was razed. Ultrasound conduction gel was applied to improve the conductivity. First, 2-D ultrasound imaging was used to visualize a cross-sectional B-mode image of the skull and brain structures of the animal. Color Doppler ultrasound was then used and the extra and intracranial arteries were indicated on the screen by two different colors (blue and red, where the blue color indicates the blood flow away from the transducer and the red color indicates the flow towards the transducer). The probe was placed above the skull and the cross-sectional scan was performed by moving the probe from back to frontal. After symmetry posterior cerebral arteries (PCA) were identified with a horizontal distance between left and right arteries was around 0.72 cm, then the probe was moved forward about 2 mm and left and right middle cerebral arteries (LMCA and RMCA) were found with the horizontal distances between left and right arteries was around 0.85 cm, and MCA are coded in red in Figure 1A. Left and right internal carotid arteries (LICA and RICA) were identified by slowly rotating the probe to the longitudinal direction and moving to lateral side of the skull; they are coded in red in Figure 1B. The probe was changed back to transactional direction and slightly moved back by 1 cm and tilted at the frontal angle; the basilar artery (BA) was localized in the middle of the skull base. It is also coded in red (Fig. 1C). Imaging depth was set at 22 mm when applying zoom. Standardized settings were used for color Doppler ultrasound: ultrasound frequency at 6.3 MHz, pulsed repetition frequency (PRF) at 4 kHz and the frame rate was 65 frames/s. Throughout the ultrasonography measurement a heating lamp was used to keep the body temperature steady to prevent hypothermia. The rats first received a baseline scan of blood flow velocity at LMCA, RMCA, LICA, RICA, and BA using spectral Doppler ultrasound. On the spectral Doppler, the color Doppler map was used to guide the placement of the pulsed Doppler gate and tracings of the arterial signal recorded. The sample volume was set for 1 mm for all the measurements and the pulsed Doppler gate was placed in the centre of the vessel. The level of wall filter was set at low for all the examinations to allow detection of vessels with low blood flow and the PRF was adjusted until the spectral waveforms were demonstrated without aliasing. The peak systolic velocity (PSV), end diastolic velocity (EDV) and time-average maximum blood flow velocity (BFV) were measured from three
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Fig. 1. Color-coded and 2-D ultrasound imaging of the cerebral arteries in the rat. (A) Left and right middle cerebral artery measured at cross-sectional direction (LMCA and RMCA, indicated by the arrow) (B) Internal carotid artery measured at longitudinal direction (ICA, indicated by the arrow) (C) Basilar artery measured at cross-sectional direction (BA, indicated by the arrow) (D) 2-D imaging to verify the filament position (E) picture of illustration of the scan position.
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consecutive Doppler spectral waveforms (Fig. 1A, B and C). The heart rate was estimated by calculating the peak-to-peak time span of the three spectral waveforms, which indicated by the crossed cursor. After 2 days rest, focal cerebral ischemia at the right side brain was induced by the intraluminal suture method (Koizumi et al. 1986; Leung et al. 2006). Briefly, the right common carotid artery was isolated from the surrounding tissue and ligated. After that, the right external carotid artery was separated and ligated. A 3–0 monofilament nylon suture, whose tip was rounded with a diameter of 0.38 0.45 mm by heating and coated with poly-l-lysine, was inserted in the right common carotid artery through a tiny cut on the vessel wall and introduced from the carotid bifurcation into the right internal carotid artery until a mild resistance was felt, with a distance around 17–20 mm from the bifurcation (Shimazu et al. 2004), thereby occluding the origin of the RMCA. The filament position could be verified by the 2-D imaging (Fig. 1D). During the occlusion of 90 min, BFV of LMCA, LICA and BA were measured. Then, reperfusion was established by gentle withdrawal of the filament and an ultrasound scan was performed at all five vessels within 30 min of the reperfusion process. The rats scored 1–2 in neurologic deficit assessment after they woke up, where score 1 means failure to extend forepaw of the affected side fully and score 2 means circling to the affected side (Longa et al. 1989). Then, post-surgery ultrasonography scan was conducted on each day from post-stroke 24 h to 96 h. The set-up of the ultrasound experiment and the position of the probe for the measurement are illustrated in Fig.1E. The body weight and behavioral test score measured with De Ryck’s scoring scale (De Ryck et al. 1989) were also recorded on a daily basis. The animal was sacrificed after the last scan on poststroke 96 h by overdose anesthesia. The brain was immediately removed from the cranium and dissected into 2 mm sections using a brain matrix (RBM-4000C; ASI Instruments Inc., Houston, Texas, USA). The brain slices were immersed in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich Co., St. Louis, Missouri, USA) in a phosphate buffered solution (PSB) at 37 C for 30 minutes. They were then fixed in 10% formaldehyde overnight. The TTC-stained sections were photographed with a digital camera and images were analyzed by software (UTHSCSA Image Tool for Windows version 3.00, San Antonio, Texas, USA). Areas of infarction were defined as those lacking red TTC stain and total infarct volumes were computed by integrating the infarct areas of sequential brain slices. All the experimental procedures were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and the experimental protocol was approved by the sub-committee ethic board of the Hong Kong Polytechnic University.
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To validate the reliability of both the ultrasound imaging technique for the cerebral blood flow velocity measurement and to check if the technique is operatordependent or not, intraclass correlation coefficient (ICC) was applied. Inter-rater reliability was evaluated by the ICC2,1 model. Two raters, separately and independently from one another, measured the basal state of blood flow velocity from LMCA, LICA, BA, RMCA and RICA of six rats under the same condition of anesthesia and recorded the results double blindly. The test-retest repeatability was evaluated by the ICC3,1 model. The same two raters separately and independently measured the BFV on the same subjects three different times with time interval between measurements was 10 min at the baseline condition before stroke. The following scales for indicating levels of reliability were applied: equal or above .90, high reliability; .80 to .89, good reliability; .70 to .79, fair reliability; and .69 or less, poor reliability (Meyers and Blesh 1962). Two-way repeated analysis of variance (ANOVA) with Bonferroni Post-hoc test was used to evaluate the BFV changes in the occlusion-reperfusion procedures and post-stroke recovery. Results are expressed as mean 6 standard deviation and the level of statistical significance is set at 0.05. RESULTS The reliability results of the ICC2,1 model showed that the Cronbach a had a range of 0.836 to 0.986 for BFV of all the five vessels, indicating that the measurement procedure was good for inter-rater reliability. The test-retest reliability results showed that the Cronbach a for the two raters had a range of 0.905 to 0.974, meaning that the intra-rater reliability was high. There was a steady state of hemodynamic values during the ultrasonography scan, which was shown from the heart rate. On the basal state, heart rate of the rats was 428 6 25 (n512) beats per minute (bpm). Occlusion of RMCA did not modify heart rate (411 6 27 bpm), neither did the reperfusion (380 6 45 bpm). During the whole recovery post-stroke stage, the heart rate was from 394 6 45 to 419 6 48 bpm, and there was no significant difference along the time. Peak systolic, end-diastolic and time-average maximum BFV at the basal state before stroke, the reperfusion and 24 h after stroke were summarized in Table 1. There was no significant difference of time-average maximum BFV between vessel pair of MCA and ICA at basal state. The baseline value of peak systolic, enddiastolic and time-average maximum BFV of RICA was 81.6 6 20.0, 46.2 6 12.3, and 58.9 6 14.3 cm/s, respectively. After release of the filament, reversed direction of cerebral blood flow was found in RICA during and after
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Table 1. Blood flow velocity (peak systolic, end diastolic and time-average maximum) of five vessels at baseline, reperfusion and 24 h post-stroke Blood flow velocity (cm/s, Mean 6 SD) Peak systolic RMCA LMCA RICA LICA BA End diastolic RMCA LMCA RICA LICA BA Time-average Maximum RMCA LMCA RICA LICA BA
Baseline
Reperfusion
24 h post-stroke
17.3 6 3.0 20.6 6 6.4 81.6 6 20.0 74.9 6 18.2 27.3 6 4.5
9.9 6 1.5* 18.6 6 6.6 250.4 6 24.6* 94.9 6 16.4 33.3 6 7.0
12.3 6 1.7** 22.0 6 5.4 267.8 6 23.0** 105.2 6 19.0** 42.8 6 6.4**
9.4 6 1.5 10.9 6 5.0 46.2 6 12.3 42.3 6 10.4 13.9 6 1.9
4.9 6 1.8* 8.3 6 3.7 223.7 6 14.8* 45.0 6 10.7 14.6 6 5.4
6.8 6 1.8 11.0 6 4.1 232.8 6 15.9** 55.9 6 15.7 23.2 6 4.4**
11.8 6 2.2 14.3 6 4.1 58.9 6 14.3 53.9 6 12.5 17.7 6 2.3
5.8 6 1.4* 10.7 6 4.7* 232.4 6 18.6* 61.1 6 12.9 20.5 6 6.6
8.5 6 1.2** 13.9 6 5.3 243.9 6 16.5** 74.6 6 12.6** 29.8 6 5.2**
RMCA 5 right middle cerebral artery; LMCA 5 left middle cerebral artery; RICA 5 right internal carotid artery; LICA 5 left internal carotid artery; BA 5 basilar artery. * p , 0.05: Baseline compared with reperfusion; ** p , 0.05: Baseline compared with 24 h post-stroke. Negative values of BFV of RICA mean the blood flow direction reversed compared with baseline condition.
the reperfusion (coded in blue, Fig. 2A). Blood flow remained in the reversed direction and the BFV remained stable along the post-stroke stage (Fig. 2 B, n 512). The time-average maximum BFV of right MCA at reperfusion and post-stroke 24–96 h (5.88.5 cm/s) was decreased significantly compared to the baseline value (11.8 6 2.2, p , 0.05) (Fig. 2C). In addition, the time-average
maximum BFV of right MCA at post-stroke 24 96 h was significantly lower compared with that of left MCA (10.714.3 cm/s, p , 0.05). Cerebral blood flow of LICA, LMCA and BA maintained the same direction during the occlusion, reperfusion and post-stroke stage. Peak systolic, end-diastolic and time-average maximum BFV at the basal state of LMCA
B
C
Fig. 2. (A) Color-coded ultrasound longitudinal imaging of right internal carotid artery (RICA) after reperfusion (B) RICA and (C) Right middle cerebral artery (RMCA) Blood flow velocity changes during the occlusion/reperfusion and along the post-stroke recovery stage. * Any significant difference between the baseline and other time of time-average maximum BFV at RMCA p , 0.05. The error bar represents 1 standard deviation (SD).
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A
B
C
Fig. 3. Blood flow velocity changes during the occlusion/reperfusion and along the post-stroke recovery stage of left middle cerebral artery (LMCA) (A), left internal carotid artery (LICA) (B) and basilar artery (BA) (C). Any significant difference between the baseline and other time of time-average maximum BFV at LMCA (*), LICA (y) and BA (z), p , 0.05, respectively. The error bar represents 1 standard deviation (SD).
were 20.6 6 6.4, 10.9 6 5.0 and 14.3 6 4.1 cm.s21. Timeaverage maximum BFV of LMCA decreased at the reperfusion state compared with those of the baseline value (p , 0.05, Fig. 3A). At LICA and BA, the peak systolic, enddiastolic and time-average maximum BFV at the basal state were 74.9 6 18.2, 42.3 6 10.4 and 53.9 6 12.5 cm.s21 and 27.3 6 4.5, 13.9 6 1.9 and 17.7 6 2.3 cm.s21, respectively. The results showed significant larger time-average maximum BFV of LICA and BA starting from post-stroke 24 h compared with the baseline value (p , 0.05) (Fig. 3B and C). The behavioral test score increased significantly (p , 0.05) from post 48 h–96 h compared with post 24 h after the MCAo/r surgery (Fig. 4). After the significant drop in the body weight after the surgery in the post-
stroke 24 h, the body weight kept stable (Fig. 4). All infarct area caused by the ischemia has been confirmed by TTC staining and the infarct volume of the 12 rats was 148 6 56 mm3. A typical brain slice picture is shown in Fig. 5. DISCUSSION In this current study, cerebral blood flow velocity of ischemia stroke rat at occlusion-reperfusion and post-stroke recovery state was evaluated through colorDoppler ultrasonography. Previous studies have shown that ultrasonography was a feasible method to measure cerebral blood flow velocity and could obtain comparable results to those obtained from some ‘‘golden standard’’
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Fig. 4. Behavioral tests results and body weight data after the surgery on a daily measured basis. Any significant difference between post 24 h and other time of behavioral score (* p , 0.05). The error bar represents 1 standard deviation (SD).
techniques, such as computed tomography (CT) angiography (Tsivgoulis et al. 2007). Tsivgoulis and coworkers found that the TCD examination yielded satisfactory agreement with urgent brain CT angiography in the evaluation of patients with acute cerebral occlusion. In addition, Els and coworkers validated the TCD measurement of cerebral blood flow velocity with the autoradiography on ischemia rabbit and found the results from ultrasonography were well correlated with those from the autoradiogram technique (Els et al. 1999). Based on previously defined categories for levels of reliability (Meyers and Blesh 1962), our ICC results demonstrated good interrater reliability and intra-rater reliability with the use of
Fig. 5. Brain slices imaging for indication of infarct area (white color).
ultrasonography to measure BFV from cerebral arteries of the rat brain. One of the findings in this study was that the reversed blood flow in RICA was found after the release of the ligament (Fig. 2A). Our finding is similar to the results of previous studies on brain hypoxia and ischemia of rat pups (Bonnin et al. 2008). Bonnin and coworkers found that the persistent occlusion of the left common carotid artery presented blood flow adaptation through the Willis circle to maintain and restore the blood flow supply in the proximal part of the left MCA and blood flow velocities were reversed in the left ICA. The anterior and posterior circulation is connected via the circle of Willis, which is formed by the unpaired anterior communicating artery (ACoA) and paired posterior communication artery. Main arteries such as the anterior cerebral artery (ACA), MCA, and PCA emerge from the circle of Willis (Uflacker 2007). In this current study, due to the permanent occlusion of RCCA and RECA, the reversed flow of RICA may be caused by the contralateral blood flow through the circle of Willis. Our results showed that BFV of RMCA at reperfusion and post-stroke stage was decreased compared with baseline value (Fig. 2C). This was in line with the previous animal studies of ischemia rabbit (Els et al. 1999) and ischemia rat (Ding et al. 2002), which both revealed a decrease blood flow in ipsilateral MCA post-surgery. Since the blood supply of ischemia area came from the contralateral side through the circle of Willis, it is reasonable that the velocity is slower than the original supply, which came directly from the main branch of ICA. In addition, this also makes the BFV of the affected side smaller compared with that of the unaffected side after stroke. The successful reflow and the decrease of the blood supply could in part be indicated from the infarct area caused from the ischemic-reperfusion lesion (Fig. 5). The white area depicted the sever damage of the occlusion-reperfusion. Taken together, all these data confirm the reflow after filament removal and revealed a dynamic blood supply redistribution following the ischemia and along the recovery stage.
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Blood flow velocity of LICA and BA were increased in the recovery stage compared with the baseline value before the surgery (Fig. 3). The higher BFV in LICA and BA compared with the baseline may indicate a compensated mechanism of blood supply to the brain at the contralateral side. Based on the behavioral score results, the rats recovered some motor function after the onset of stroke (Fig. 4). Previous clinical and animal studies have indicated that the cortical ischemia leads to functional impairments that substantially improve during the early period after the insult (Horgan and Finn 1997; Metz et al. 2005; Whishaw 2000). For example, Metz and coworkers found that early postoperative performance improvements of ischemia rat indicated compensatory mechanisms since the animal developed alternative movement strategies to successfully carry out the tasks. Our results might help to explain the reasons of the recovery based on the adaptive changes of blood supply from the contralateral hemisphere to compensate the ipsilateral side after the onset of stroke. There is a limitation of this current study that should be noticed. It is the lack of consistent measures of blood pressure during the measurement. Although the stable heart rate may indicate a reliable cardio output during the whole measurement, it should be concerned to avoid the large variation of the vascular dilation and hypotension during the surgery and after stroke. However, caution is needed when obtaining the blood pressure because any restraint for the purpose of measurement could interact with occlusion-reperfusion to confound results. Our study demonstrates the reliability of measuring the cerebral blood flow velocity of ischemia stroke rat by using ultrasound imaging and this technique might help to elucidate the surgical procedures. The detailed information of the blood flow changes helps to characterize the dramatic cerebral hemodynamic changes occurring during cerebral ischemia and reperfusion, as well as the variability of reflow. Further study of investigating the correlation between blood flow redistribution and the neuroprotective treatment after ischemia stoke is warranted, which could provide better understanding of the recovery mechanism after stroke. Acknowledgements—This study was supported by the Postdoctoral Fellowship Scheme, The Hong Kong Polytechnic University (G-YX1D).
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Original Contribution EVALUATION OF CEREBRAL BLOOD FLOW CHANGES IN FOCAL CEREBRAL ISCHEMIA RATS BY USING TRANSCRANIAL DOPPLER ULTRASONOGRAPHY LE LI, ZHENG KE, KAI YU TONG and MICHAEL YING Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong SAR, China (Received 12 May 2009; revised 31 December 2009; in final form 15 January 2010)
Abstract—Ischemic stroke is typically characterized by the disruption of cerebral blood flow. This study aimed to consecutively evaluate the cerebral blood flow changes in a focal ischemia rat model during the occlusionreperfusion procedure and along the recovery stage after stroke. In 12 Sprague Dawley (SD) rats, a middle cerebral artery occlusion/reperfusion (MCAo/r) surgery was conducted, which combines a permanent occlusion of the right common carotid artery (CCA), external carotid artery (ECA) and a transient occlusion of the right internal carotid artery (ICA) and middle cerebral artery (MCA) with a monofilament introduced from the proximal ICA towards the distal right ICA then removed after 90 min. Blood flow velocity (BFV) from the concerned arteries were measured using ultrasonography (13–4 MHz) at the basal stage before the surgery, after the reperfusion stage and during the post-stroke status. At reperfusion stage and after, BFV increased significantly in the left ICA and in the basilar artery (BA) (starting from post-24 h, p , 0.05 vs. basal). Moreover, BFV were reversed in the distal right ICA and reflow was recorded in the right MCA. Time-average maximum BFV in the right MCA at reperfusion and post-stroke 24–96 h was decreased significantly (p , 0.05 vs. basal). The reversed flow in the right ICA was enabled by the settlement of the collateral supply through the circle of Willis which consisted in higher BFV in the opposite ICA and in the BA still 24 h, although the proximal right ICA remain occluded. Ultrasound measurement of BFV helps to provide information on the redistribution of the blood flow supply after the onset of stroke. (E-mail: [email protected]) Ó 2010 World Federation for Ultrasound in Medicine & Biology. Key Words: Blood flow velocity, Focal cerebral ischemia, Rat, Ultrasonography.
perfusion, helping to describe the ischemic area and the surrounding penumbra. However, a consecutive examination of the cerebral hemodynamic during acute and subacute stroke is not possible with these techniques due to their insufficient temporal resolution and restricted access. Laser-Doppler flowmetry was used to monitor ipsilateral or local cerebral blood flow for occlusion and reperfusion in some animal studies (Ding et al. 2002; Sayeed et al. 2006). However, to increase the penetrability, sometimes the skull of the animal needed to be thinned that might cause unnecessary damage to the animal (Burnett et al. 2006). Transcranial Doppler (TCD) ultrasonographic assessment has been widely applied in the cerebral ischemia detection and evaluation. After Aaslid et al. (1982) introduced a 2 MHz Doppler system that allowed adequate penetration through the intact skull and demonstrated clinical useful recordings of blood flow velocities of the intracranial blood vessels, this noninvasive, reproducible and relatively inexpensive testing has become routine in the clinical management of the cerebral blood flow of patients with cerebrovascular disease or subarachnoid hemorrhage
INTRODUCTION Ischemic stroke happens when there is inadequate delivery of glucose and oxygen to the brain, which is caused by a critical reduction of cerebral blood flow (CBF), mostly due to the occlusion or stenosis of a brain-supplying vessel (Baron 1999; Choy et al. 2006). The reduction of cerebral flow results in energy failure and secondary biochemical disturbance, eliciting a robust inflammatory response in situ and this inflammatory process contributes to the development of cell injury in stroke (Dirnagl et al. 1999). Therefore, it is desirable to obtain information of blood flow changes from the actual cerebral perfusion deficit and on its time-course in patients suffering from stroke. Positron-emission tomography (PET) (Heiss and Podreka 1993) and magnetic resonance imaging (MRI) techniques (Besselmann et al. 2001; Yang et al. 2008) were used to evaluate cerebral diffusion and
Address correspondence to: Dr. Kai yu Tong, Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China. E-mail: [email protected] 595
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(Rigamonti et al. 2008). Some important and widespread applications of TCD have been reported in the detection of stenosis in the basal intracranial arteries (Demchuk et al. 2000); evaluation of extracranial internal carotid artery disease (Wilterdink et al. 1997), monitoring in cerebral embolism (Padayachee et al. 1986); and in the diagnosis of cerebral vasospasm (Proust et al. 1999), etc. However, animal studies are warranted if the mechanism and physiologic bases of ischemic stroke needed to be evaluated and these could help for a better understanding of the disease and better planning for neuroprotective treatment. The mainstream animal model of focal ischemic stroke is the middle cerebral artery occlusion/reperfusion (MCAo/r). In addition, the most conventional technique is the intraluminal model (Koizumi et al. 1999; Leung et al. 2006; Shimazu et al. 2004). However, this surgical procedure has a limitation in that the occlusion of the vessel could only be judged from experience and requires considerable skills. Although the markers on the filament could provide information on the depth of the insertion, the variation of individual animal anatomical structure could greatly affect the successful rate of the surgery. Modern ultrasound units and transducers with a smaller sample volume and an optimized focus allow measurements in small animal vessels with a high temporal resolution (Bonnin et al. 2008; Els et al. 1999). Therefore, two-dimensional (2-D) ultrasonography could help to illustrate the brain anatomy and make sure the filament is in the right position for improving the success rate of the model. The ultrasonographic measurement allows analysis of rapid changes in blood flow as early as arterial occlusion is performed and the evaluation could be repeated for a longitudinal survey at different time points. Ultrasound imaging on ischemic brain based on animal models has been reported on rabbit (Els et al. 1999) and rat pups (Bonnin et al. 2008). However, the detailed information of CBF velocity changes at different lesion time and the subacute stage (i.e., 16–24 h after surgery) as well as at the chronic stroke stage (160–168 h post-surgery, SoltanianZadeh et al. 2003) is not clear yet. In this current study, cerebral blood flow changes occurring during the whole procedure of cerebral ischemia-reperfusion and along the 4 days post-stroke recovery period from a focal ischemia rat model were evaluated by using highfrequency color Doppler ultrasonography, which may help to provide more information on the redistribution of blood flow supply after the onset of stroke. MATERIALS AND METHODS Twelve young male Sprague Dawley (SD) rats with body weight around 230–250 g were selected and housed individually during a 12 h light/dark cycle with access to
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food and water ad libitum in a temperature-controlled environment (20–22 C). Animals were anaesthetized with chloral hydrate (0.4 mg/kg, intraperitoneal) and all ultrasound assessments were conducted with an Esaote MyLab 70 X-view ultrasound unit in conjunction with a 13 to 4 MHz linear transducer (Esaote, Genova, Italy). The rat was placed on a stereotaxic frame and the probe was put on the skull of the rat after the fur was razed. Ultrasound conduction gel was applied to improve the conductivity. First, 2-D ultrasound imaging was used to visualize a cross-sectional B-mode image of the skull and brain structures of the animal. Color Doppler ultrasound was then used and the extra and intracranial arteries were indicated on the screen by two different colors (blue and red, where the blue color indicates the blood flow away from the transducer and the red color indicates the flow towards the transducer). The probe was placed above the skull and the cross-sectional scan was performed by moving the probe from back to frontal. After symmetry posterior cerebral arteries (PCA) were identified with a horizontal distance between left and right arteries was around 0.72 cm, then the probe was moved forward about 2 mm and left and right middle cerebral arteries (LMCA and RMCA) were found with the horizontal distances between left and right arteries was around 0.85 cm, and MCA are coded in red in Figure 1A. Left and right internal carotid arteries (LICA and RICA) were identified by slowly rotating the probe to the longitudinal direction and moving to lateral side of the skull; they are coded in red in Figure 1B. The probe was changed back to transactional direction and slightly moved back by 1 cm and tilted at the frontal angle; the basilar artery (BA) was localized in the middle of the skull base. It is also coded in red (Fig. 1C). Imaging depth was set at 22 mm when applying zoom. Standardized settings were used for color Doppler ultrasound: ultrasound frequency at 6.3 MHz, pulsed repetition frequency (PRF) at 4 kHz and the frame rate was 65 frames/s. Throughout the ultrasonography measurement a heating lamp was used to keep the body temperature steady to prevent hypothermia. The rats first received a baseline scan of blood flow velocity at LMCA, RMCA, LICA, RICA, and BA using spectral Doppler ultrasound. On the spectral Doppler, the color Doppler map was used to guide the placement of the pulsed Doppler gate and tracings of the arterial signal recorded. The sample volume was set for 1 mm for all the measurements and the pulsed Doppler gate was placed in the centre of the vessel. The level of wall filter was set at low for all the examinations to allow detection of vessels with low blood flow and the PRF was adjusted until the spectral waveforms were demonstrated without aliasing. The peak systolic velocity (PSV), end diastolic velocity (EDV) and time-average maximum blood flow velocity (BFV) were measured from three
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Fig. 1. Color-coded and 2-D ultrasound imaging of the cerebral arteries in the rat. (A) Left and right middle cerebral artery measured at cross-sectional direction (LMCA and RMCA, indicated by the arrow) (B) Internal carotid artery measured at longitudinal direction (ICA, indicated by the arrow) (C) Basilar artery measured at cross-sectional direction (BA, indicated by the arrow) (D) 2-D imaging to verify the filament position (E) picture of illustration of the scan position.
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consecutive Doppler spectral waveforms (Fig. 1A, B and C). The heart rate was estimated by calculating the peak-to-peak time span of the three spectral waveforms, which indicated by the crossed cursor. After 2 days rest, focal cerebral ischemia at the right side brain was induced by the intraluminal suture method (Koizumi et al. 1986; Leung et al. 2006). Briefly, the right common carotid artery was isolated from the surrounding tissue and ligated. After that, the right external carotid artery was separated and ligated. A 3–0 monofilament nylon suture, whose tip was rounded with a diameter of 0.38 0.45 mm by heating and coated with poly-l-lysine, was inserted in the right common carotid artery through a tiny cut on the vessel wall and introduced from the carotid bifurcation into the right internal carotid artery until a mild resistance was felt, with a distance around 17–20 mm from the bifurcation (Shimazu et al. 2004), thereby occluding the origin of the RMCA. The filament position could be verified by the 2-D imaging (Fig. 1D). During the occlusion of 90 min, BFV of LMCA, LICA and BA were measured. Then, reperfusion was established by gentle withdrawal of the filament and an ultrasound scan was performed at all five vessels within 30 min of the reperfusion process. The rats scored 1–2 in neurologic deficit assessment after they woke up, where score 1 means failure to extend forepaw of the affected side fully and score 2 means circling to the affected side (Longa et al. 1989). Then, post-surgery ultrasonography scan was conducted on each day from post-stroke 24 h to 96 h. The set-up of the ultrasound experiment and the position of the probe for the measurement are illustrated in Fig.1E. The body weight and behavioral test score measured with De Ryck’s scoring scale (De Ryck et al. 1989) were also recorded on a daily basis. The animal was sacrificed after the last scan on poststroke 96 h by overdose anesthesia. The brain was immediately removed from the cranium and dissected into 2 mm sections using a brain matrix (RBM-4000C; ASI Instruments Inc., Houston, Texas, USA). The brain slices were immersed in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich Co., St. Louis, Missouri, USA) in a phosphate buffered solution (PSB) at 37 C for 30 minutes. They were then fixed in 10% formaldehyde overnight. The TTC-stained sections were photographed with a digital camera and images were analyzed by software (UTHSCSA Image Tool for Windows version 3.00, San Antonio, Texas, USA). Areas of infarction were defined as those lacking red TTC stain and total infarct volumes were computed by integrating the infarct areas of sequential brain slices. All the experimental procedures were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and the experimental protocol was approved by the sub-committee ethic board of the Hong Kong Polytechnic University.
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To validate the reliability of both the ultrasound imaging technique for the cerebral blood flow velocity measurement and to check if the technique is operatordependent or not, intraclass correlation coefficient (ICC) was applied. Inter-rater reliability was evaluated by the ICC2,1 model. Two raters, separately and independently from one another, measured the basal state of blood flow velocity from LMCA, LICA, BA, RMCA and RICA of six rats under the same condition of anesthesia and recorded the results double blindly. The test-retest repeatability was evaluated by the ICC3,1 model. The same two raters separately and independently measured the BFV on the same subjects three different times with time interval between measurements was 10 min at the baseline condition before stroke. The following scales for indicating levels of reliability were applied: equal or above .90, high reliability; .80 to .89, good reliability; .70 to .79, fair reliability; and .69 or less, poor reliability (Meyers and Blesh 1962). Two-way repeated analysis of variance (ANOVA) with Bonferroni Post-hoc test was used to evaluate the BFV changes in the occlusion-reperfusion procedures and post-stroke recovery. Results are expressed as mean 6 standard deviation and the level of statistical significance is set at 0.05. RESULTS The reliability results of the ICC2,1 model showed that the Cronbach a had a range of 0.836 to 0.986 for BFV of all the five vessels, indicating that the measurement procedure was good for inter-rater reliability. The test-retest reliability results showed that the Cronbach a for the two raters had a range of 0.905 to 0.974, meaning that the intra-rater reliability was high. There was a steady state of hemodynamic values during the ultrasonography scan, which was shown from the heart rate. On the basal state, heart rate of the rats was 428 6 25 (n512) beats per minute (bpm). Occlusion of RMCA did not modify heart rate (411 6 27 bpm), neither did the reperfusion (380 6 45 bpm). During the whole recovery post-stroke stage, the heart rate was from 394 6 45 to 419 6 48 bpm, and there was no significant difference along the time. Peak systolic, end-diastolic and time-average maximum BFV at the basal state before stroke, the reperfusion and 24 h after stroke were summarized in Table 1. There was no significant difference of time-average maximum BFV between vessel pair of MCA and ICA at basal state. The baseline value of peak systolic, enddiastolic and time-average maximum BFV of RICA was 81.6 6 20.0, 46.2 6 12.3, and 58.9 6 14.3 cm/s, respectively. After release of the filament, reversed direction of cerebral blood flow was found in RICA during and after
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Table 1. Blood flow velocity (peak systolic, end diastolic and time-average maximum) of five vessels at baseline, reperfusion and 24 h post-stroke Blood flow velocity (cm/s, Mean 6 SD) Peak systolic RMCA LMCA RICA LICA BA End diastolic RMCA LMCA RICA LICA BA Time-average Maximum RMCA LMCA RICA LICA BA
Baseline
Reperfusion
24 h post-stroke
17.3 6 3.0 20.6 6 6.4 81.6 6 20.0 74.9 6 18.2 27.3 6 4.5
9.9 6 1.5* 18.6 6 6.6 250.4 6 24.6* 94.9 6 16.4 33.3 6 7.0
12.3 6 1.7** 22.0 6 5.4 267.8 6 23.0** 105.2 6 19.0** 42.8 6 6.4**
9.4 6 1.5 10.9 6 5.0 46.2 6 12.3 42.3 6 10.4 13.9 6 1.9
4.9 6 1.8* 8.3 6 3.7 223.7 6 14.8* 45.0 6 10.7 14.6 6 5.4
6.8 6 1.8 11.0 6 4.1 232.8 6 15.9** 55.9 6 15.7 23.2 6 4.4**
11.8 6 2.2 14.3 6 4.1 58.9 6 14.3 53.9 6 12.5 17.7 6 2.3
5.8 6 1.4* 10.7 6 4.7* 232.4 6 18.6* 61.1 6 12.9 20.5 6 6.6
8.5 6 1.2** 13.9 6 5.3 243.9 6 16.5** 74.6 6 12.6** 29.8 6 5.2**
RMCA 5 right middle cerebral artery; LMCA 5 left middle cerebral artery; RICA 5 right internal carotid artery; LICA 5 left internal carotid artery; BA 5 basilar artery. * p , 0.05: Baseline compared with reperfusion; ** p , 0.05: Baseline compared with 24 h post-stroke. Negative values of BFV of RICA mean the blood flow direction reversed compared with baseline condition.
the reperfusion (coded in blue, Fig. 2A). Blood flow remained in the reversed direction and the BFV remained stable along the post-stroke stage (Fig. 2 B, n 512). The time-average maximum BFV of right MCA at reperfusion and post-stroke 24–96 h (5.88.5 cm/s) was decreased significantly compared to the baseline value (11.8 6 2.2, p , 0.05) (Fig. 2C). In addition, the time-average
maximum BFV of right MCA at post-stroke 24 96 h was significantly lower compared with that of left MCA (10.714.3 cm/s, p , 0.05). Cerebral blood flow of LICA, LMCA and BA maintained the same direction during the occlusion, reperfusion and post-stroke stage. Peak systolic, end-diastolic and time-average maximum BFV at the basal state of LMCA
B
C
Fig. 2. (A) Color-coded ultrasound longitudinal imaging of right internal carotid artery (RICA) after reperfusion (B) RICA and (C) Right middle cerebral artery (RMCA) Blood flow velocity changes during the occlusion/reperfusion and along the post-stroke recovery stage. * Any significant difference between the baseline and other time of time-average maximum BFV at RMCA p , 0.05. The error bar represents 1 standard deviation (SD).
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A
B
C
Fig. 3. Blood flow velocity changes during the occlusion/reperfusion and along the post-stroke recovery stage of left middle cerebral artery (LMCA) (A), left internal carotid artery (LICA) (B) and basilar artery (BA) (C). Any significant difference between the baseline and other time of time-average maximum BFV at LMCA (*), LICA (y) and BA (z), p , 0.05, respectively. The error bar represents 1 standard deviation (SD).
were 20.6 6 6.4, 10.9 6 5.0 and 14.3 6 4.1 cm.s21. Timeaverage maximum BFV of LMCA decreased at the reperfusion state compared with those of the baseline value (p , 0.05, Fig. 3A). At LICA and BA, the peak systolic, enddiastolic and time-average maximum BFV at the basal state were 74.9 6 18.2, 42.3 6 10.4 and 53.9 6 12.5 cm.s21 and 27.3 6 4.5, 13.9 6 1.9 and 17.7 6 2.3 cm.s21, respectively. The results showed significant larger time-average maximum BFV of LICA and BA starting from post-stroke 24 h compared with the baseline value (p , 0.05) (Fig. 3B and C). The behavioral test score increased significantly (p , 0.05) from post 48 h–96 h compared with post 24 h after the MCAo/r surgery (Fig. 4). After the significant drop in the body weight after the surgery in the post-
stroke 24 h, the body weight kept stable (Fig. 4). All infarct area caused by the ischemia has been confirmed by TTC staining and the infarct volume of the 12 rats was 148 6 56 mm3. A typical brain slice picture is shown in Fig. 5. DISCUSSION In this current study, cerebral blood flow velocity of ischemia stroke rat at occlusion-reperfusion and post-stroke recovery state was evaluated through colorDoppler ultrasonography. Previous studies have shown that ultrasonography was a feasible method to measure cerebral blood flow velocity and could obtain comparable results to those obtained from some ‘‘golden standard’’
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Fig. 4. Behavioral tests results and body weight data after the surgery on a daily measured basis. Any significant difference between post 24 h and other time of behavioral score (* p , 0.05). The error bar represents 1 standard deviation (SD).
techniques, such as computed tomography (CT) angiography (Tsivgoulis et al. 2007). Tsivgoulis and coworkers found that the TCD examination yielded satisfactory agreement with urgent brain CT angiography in the evaluation of patients with acute cerebral occlusion. In addition, Els and coworkers validated the TCD measurement of cerebral blood flow velocity with the autoradiography on ischemia rabbit and found the results from ultrasonography were well correlated with those from the autoradiogram technique (Els et al. 1999). Based on previously defined categories for levels of reliability (Meyers and Blesh 1962), our ICC results demonstrated good interrater reliability and intra-rater reliability with the use of
Fig. 5. Brain slices imaging for indication of infarct area (white color).
ultrasonography to measure BFV from cerebral arteries of the rat brain. One of the findings in this study was that the reversed blood flow in RICA was found after the release of the ligament (Fig. 2A). Our finding is similar to the results of previous studies on brain hypoxia and ischemia of rat pups (Bonnin et al. 2008). Bonnin and coworkers found that the persistent occlusion of the left common carotid artery presented blood flow adaptation through the Willis circle to maintain and restore the blood flow supply in the proximal part of the left MCA and blood flow velocities were reversed in the left ICA. The anterior and posterior circulation is connected via the circle of Willis, which is formed by the unpaired anterior communicating artery (ACoA) and paired posterior communication artery. Main arteries such as the anterior cerebral artery (ACA), MCA, and PCA emerge from the circle of Willis (Uflacker 2007). In this current study, due to the permanent occlusion of RCCA and RECA, the reversed flow of RICA may be caused by the contralateral blood flow through the circle of Willis. Our results showed that BFV of RMCA at reperfusion and post-stroke stage was decreased compared with baseline value (Fig. 2C). This was in line with the previous animal studies of ischemia rabbit (Els et al. 1999) and ischemia rat (Ding et al. 2002), which both revealed a decrease blood flow in ipsilateral MCA post-surgery. Since the blood supply of ischemia area came from the contralateral side through the circle of Willis, it is reasonable that the velocity is slower than the original supply, which came directly from the main branch of ICA. In addition, this also makes the BFV of the affected side smaller compared with that of the unaffected side after stroke. The successful reflow and the decrease of the blood supply could in part be indicated from the infarct area caused from the ischemic-reperfusion lesion (Fig. 5). The white area depicted the sever damage of the occlusion-reperfusion. Taken together, all these data confirm the reflow after filament removal and revealed a dynamic blood supply redistribution following the ischemia and along the recovery stage.
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Blood flow velocity of LICA and BA were increased in the recovery stage compared with the baseline value before the surgery (Fig. 3). The higher BFV in LICA and BA compared with the baseline may indicate a compensated mechanism of blood supply to the brain at the contralateral side. Based on the behavioral score results, the rats recovered some motor function after the onset of stroke (Fig. 4). Previous clinical and animal studies have indicated that the cortical ischemia leads to functional impairments that substantially improve during the early period after the insult (Horgan and Finn 1997; Metz et al. 2005; Whishaw 2000). For example, Metz and coworkers found that early postoperative performance improvements of ischemia rat indicated compensatory mechanisms since the animal developed alternative movement strategies to successfully carry out the tasks. Our results might help to explain the reasons of the recovery based on the adaptive changes of blood supply from the contralateral hemisphere to compensate the ipsilateral side after the onset of stroke. There is a limitation of this current study that should be noticed. It is the lack of consistent measures of blood pressure during the measurement. Although the stable heart rate may indicate a reliable cardio output during the whole measurement, it should be concerned to avoid the large variation of the vascular dilation and hypotension during the surgery and after stroke. However, caution is needed when obtaining the blood pressure because any restraint for the purpose of measurement could interact with occlusion-reperfusion to confound results. Our study demonstrates the reliability of measuring the cerebral blood flow velocity of ischemia stroke rat by using ultrasound imaging and this technique might help to elucidate the surgical procedures. The detailed information of the blood flow changes helps to characterize the dramatic cerebral hemodynamic changes occurring during cerebral ischemia and reperfusion, as well as the variability of reflow. Further study of investigating the correlation between blood flow redistribution and the neuroprotective treatment after ischemia stoke is warranted, which could provide better understanding of the recovery mechanism after stroke. Acknowledgements—This study was supported by the Postdoctoral Fellowship Scheme, The Hong Kong Polytechnic University (G-YX1D).
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