Perfusion Imaging of the Brain by B-Mode Ultrasonography
An Experimental Study in Rabbits
Background and Purpose The purpose of this study was to assess the ultrasonic imaging of normal rabbit brain by using echocontrast enhancer to identify the brain tissue perfusion.
Methods A solution of SH/TA-508 (containing microbubbles of mean size 2 to 4 μm; n=4) was used for contrast enhancement of rabbit brain tissue and was administered through the left internal carotid artery. Contrast enhancement in the ipsilateral parietal area was examined by placing a 7.5-MHz transducer on the dura after a craniectomy. Throughout the experiment, we measured parameters including arterial blood pressure, arterial blood gases, and serum glucose and hematocrit levels. We investigated whether the administration of an echocontrast enhancer would be safe for the brain tissue (n=4).
Results After injection of 100 mg/mL SH/TA-508, contrast enhancement was clearly observed within approximately 1 second in the left hemisphere (especially in the left cerebral cortex) in all animals and disappeared within 3 seconds after the end of injection. All physiological parameters remained within the normal range throughout the experiment. No focal abnormalities were observed on hematoxylin-eosin staining of the brain tissue.
Conclusions Contrast enhancement was observed in the ipsilateral hemisphere with the administration of SH/TA-508. Contrast enhancement on B-mode brain imaging would be useful for real-time identification of brain tissue perfusion and should become a promising method for bedside clinical assessment of brain tissue perfusion.
Real-time identification of the degree of perfusion in damaged brain tissue can be useful for assessing the prognosis of acute ischemic cerebrovascular diseases. The ultrasonic method that reveals several physiological and pathological real-time conditions is one such modality. Ito et al1 reported that simultaneous regional wall motion and myocardial perfusion can be performed in patients with acute myocardial infarction with myocardial contrast echocardiography. Transcranial B-mode real-time duplex sonography was recently used successfully to assess the development and resolution of posthemorrhagic hydrocephalus in newborn infants2 and the temporal changes of intracranial hemorrhage in adults.3 Transcranial color-coded duplex sonography with intravenous injection of an echocontrast enhancer (SH U 508 A)4 gradually has become an accepted method of visualizing the intracranial vessels; the peripheral branches of the anterior, middle, and posterior cerebral arteries; and the deep cerebral veins.
Additional experimental study is needed before the ultrasonic method is used clinically for brain tissue perfusion imaging. Our objective was to document the ultrasonic imaging of contrast enhancement in normal rabbit brain tissue by using intracarotid injection of a new contrast medium, SH/TA-508 (SH U 508 A), to identify the degree of perfusion. We also sought to determine whether intracarotid injection of SH/TA-508 would produce focal abnormalities in rabbit brain tissue.
Materials and Methods
Japanese White rabbits (body weight, 3 to 3.5 kg) were used for this study. Each animal was anesthetized with an intravenous injection of urethane (1200 mg/kg). The left common, internal, and external carotid arteries were exposed and isolated with standard microsurgical techniques. Intracarotid drug administration was performed using an 18-gauge angiocatheter introduced retrogradely into the main trunk of the left external carotid artery and secured with a silk ligature.4
With the rabbit placed in a prone position, B-mode imaging of the brain tissue was performed with a 7.5-MHz transducer (EUB-450, Hitachi Medico). Assessment of the intracranial structure was impossible by transcranial B-mode imaging when the probe was placed on the intact head, so excision of the skin, subcutaneous tissue, and muscle covering the bilateral parietal area was necessary. A craniectomy was performed to expose most of the bilateral hemisphere, with the dura mater kept intact. With the probe placed on the dura, the intracranial structure was assessed in each rabbit. Imaging was initiated 10 seconds before the injection of the contrast enhancer and was continued until the disappearance of the film contrast. Serial images were recorded on videotape with a VHS recorder.
The echocontrast enhancer (SH/TA-508; Schering AG) that we used contained a saccharide-stabilized medium consisting of galactose (99.9%) and palmitic acid (0.1%). Saccharide-stabilized microbubbles with a mean size of 2 to 4 μm were suspended in a carrier medium, a previously reported promising new approach to enhance the echocontrast.6 After SH/TA-508 and distilled water were mixed for several seconds, the solution was slowly injected after 2 minutes (up to 0.5 mL/5 s) from the left external carotid artery catheter (n=4).
We then examined whether the intracarotid administration of SH/TA-508 damaged the brain tissue. Each rabbit was placed in a supine position. A catheter was placed in the left femoral artery for manometric monitoring of mean arterial blood pressure. A right femoral artery was cannulated with an 18-gauge angiocatheter, and arterial blood gases (pH, Pao2, and Paco2) were monitored through the right arterial line. A right femoral vein was cannulated with an 18-gauge angiocatheter, and hematocrit and blood glucose levels were determined by the centrifugation method and automated enzymatic method, respectively. Normothermia was maintained with an adjustable heating blanket. Slow bolus injection of SH/TA-508 (up to 2 mL/20 s; four times the amount of the above study) was performed from the left external carotid artery catheter (n=4). Arterial blood gases and levels of serum glucose and hematocrit were examined four times: before the injection, immediately after it, and at 10 and 60 minutes. Catheters were removed after the physiological parameters were measured.
Seven days after the injection of SH/TA-508, the rabbits were killed with an overdose of intra-arterial urethane for neuropathological investigation. A large midline thoracotomy was performed immediately, and the ascending aorta was cannulated with a 16-gauge angiocatheter through the left ventricle.7 The descending aorta was clamped, and the right atrium was opened. Transcardiac perfusion was carried out through the left ventricle first with 500 mL of 0.9% saline to displace blood from the patent vessels and second with 500 mL of 10% neutralized formalin at a pressure of 120 mm Hg. The brain was then removed, fixed, and cut in bread-loaf fashion into 2-mm slices. Sections were stained with hematoxylin-eosin.
Physiological parameters were compared using one-way ANOVA, with Dunnett’s test for multiple comparisons between groups. Differences were considered to be statistically significant at values of P<.05.
Contrast enhancement was observed in the ipsilateral left whole hemisphere in all rabbits after the injection of 200 mg/mL (Fig 1⇓) and 150 mg/mL SH/TA-508. This contrast enhancement persisted for more than 10 seconds (mean: 200 mg/mL, 42 seconds; 150 mg/mL, 18 seconds) after the end of the injection. After injection of a lower concentration (100 mg/mL) of SH/TA-508, contrast enhancement was also observed in the left hemisphere (Fig 2⇓), especially in the left cerebral cortex, in all animals. Contrast enhancement was observed in the left cerebral cortex and in a portion of the basal ganglia in 2 rabbits, in portions of both the cerebral cortex and the basal ganglia in 1, and in only the cerebral cortex in 1. With 100 mg/mL SH/TA-508, contrast enhancement was observed within approximately 1 second after the start of injection and disappeared within 3 seconds after the end of injection. No residual contrast enhancement was observed more than 5 seconds after the end of injection in any animal.
With the injection of SH/TA-508 (concentration, 100 mg/mL), mean arterial blood pressure, arterial blood gases (pH, Pao2, and Paco2), and levels of serum glucose and hematocrit were maintained within normal range throughout the experiment (Table⇓). There were no significant differences in physiological parameters among the rabbits. No neurological or physical abnormalities were detected for 7 days after the injection of SH/TA-508, and no focal neuropathological abnormality was seen in rabbit brain tissue with hematoxylin-eosin staining of the supratentorial gray matter or basal ganglia. No anoxic lesions were detected in the left cerebral cortex (Fig 3⇓).
The real-time identification of perfusion in damaged brain tissue is useful for assessing patient prognosis. It is reported in describing the “no-reflow phenomenon”8 that even a short ischemic period that does not destroy parenchymal cells per se is indirectly lethal to these cells in rabbits. Both narrowing of the capillary lumina and the concentration of macromolecules and formed elements in the blood result in an impaired flow through the microvasculature when the ischemia is terminated. It was recently demonstrated that myocardial contrast echocardiography could assess the patterns of myocardial perfusion after successful reflow in patients with acute myocardial infarction.1 Early thrombolytic therapy with recombinant tissue plasminogen activator in patients with acute ischemic stroke was cited as an attractive approach to treating acute focal cerebral ischemia.9 10 Therefore, if contrast enhancement of brain tissue in B-mode imaging can be safely performed, the brain tissue perfusion caused by acute ischemic cerebrovascular diseases immediately after early thrombolytic therapy can be identified and/or monitored during cerebral angiography.
There are two important safety concerns to consider during the injection of a contrast enhancer: the toxicity of the microbubbles and the toxicity of the carrier medium. The air microbubbles in echocontrast preparations must be less than 10 μm in diameter to prevent their being trapped in the capillary vasculature and to avoid the risk of transient but significant toxicity.6 The SH/TA-508 consists of uniform microbubbles less than 10 μm in diameter (approximately 24 μm). In our study, all physiological parameters remained within the normal range throughout the experiment. In cases of arterial air embolism after open-heart surgery, cerebral dysfunction may occur in subtentorial structures, but involvement of the supratentorial gray matter in the distribution of cerebral major arteries and anoxic damage in the hippocampal formation are more common.11 12 No focal abnormalities were seen in rabbit brain tissue with hematoxylin-eosin staining. These data indicate that the contrast enhancer SH/TA-508 can be used safely with ultrasound brain imaging.
After the injection of 100 mg/mL SH/TA-508, contrast enhancement was observed in the left hemisphere, especially in the left cerebral cortex; perhaps this is because the ipsilateral regional cerebral blood flow in the cerebral cortex from one side of the internal carotid artery might be slightly greater than that in the basal ganglia and because the microbubbles of the SH/TA-508 were unstable at a concentration of 100 mg/mL. By use of higher concentrations of SH/TA-508 (200 mg/mL in intravenous phase-two study4 ), significant contrast enhancement was obtained within the left whole hemisphere that persisted for more than 10 seconds after the end of the injection. This prolongation may be attributable to an incomplete dissolution of the contrast medium. In cerebral angiograms, the circulation time for a normal adult is 4.39±0.94 seconds.13 To ensure safety, we used a lower concentration of the SH/TA-508 (100 mg/mL) in the subsequent contrast enhancement study of the brain tissue.
In the present study, the whole rabbit brains (body weight, 3 to 3.5 kg) weighed 12 to 14 g (0.4%).14 The mean regional cerebral blood flow was previously reported to be approximately 0.6 mL/g per minute.15 The cerebral blood flow per second in the rabbit hemisphere is estimated to be 0.06 to 0.07 mL. Therefore, the calculated blood flow per second in one side of the internal carotid artery would be around 0.07 mL. An infusion of SH/TA-508 of 0.07 mL/s of the rabbit brains in contrast enhancement was not apparent in the left hemisphere in our preliminary study, so we administered the infusion at 0.1 mL/s. Contrast enhancement was clearly observed in the ipsilateral hemisphere at this rate, and no side effects were noted during the experiment.
Sonicated albumin microbubbles (mean size, 5 μm16 ) are frequently used in myocardial contrast echocardiography. After the injection of sonicated albumin microbubbles by the same method in our study, we observed slight contrast enhancement in only a portion of the left hemisphere in 2 of 4 rabbits (Fig 4⇓), possibly because of the fragility of the microbubbles. After the injection of human albumin solution without sonication, contrast enhancement was not observed in any rabbit. Our findings suggest that contrast enhancement of human brain tissue could be used with the ultrasonic imaging method if the microbubbles (mean size, 5 μm) and the carrier medium were nontoxic and safe for clinical use.
Assessment of the intracranial structure by transcranial B-mode imaging was impossible in rabbits with an intact calvarium. Applied ultrasound energy of high resolution (7.5 MHz) may be absorbed and scattered by the skull. However, transcranial B-mode (2.5 MHz) real-time duplex sonography has recently been used safely and effectively to detect parenchymal lesions in adult patients.3 In addition, intraoperative real-time ultrasonography has been used neurosurgically to identify deep lesions and to provide information about their solid and cystic components.17 Contrast enhancement should be useful in assessing brain tissue perfusion if the contrast enhancer administered via intracarotid injection is verified to be safe for clinical use.
This study was supported in part by research grants for the cerebrovascular diseases from the Ministry of Health and Welfare and the Smoking Research Foundation, Japan. We are grateful to M. Shimomura and M. Tsunoda for their secretarial assistance.
- Received June 1, 1995.
- Accepted August 30, 1995.
- Copyright © 1995 by American Heart Association
Ito H, Tomooka T, Sakai N, Yu H, Higashino Y, Fujii K, Masuyama T, Kitabatake A, Minamino T. Lack of myocardial perfusion immediately after successful thrombolysis: a predictor of poor recovery of left ventricular function in anterior myocardial infarction. Circulation. 1992;85:1699-1705.
Seidel G, Kaps M, Dorndorf W. Transcranial color-coded duplex sonography of intracerebral hematomas in adults. Stroke. 1993;24:1519-1527.
Bogdahn U, Becker G, Schlief R, Reddig J, Hassel W. Contrast-enhanced transcranial color-coded real-time sonography: results of a phase-two study. Stroke. 1993;24:676-684.
Hamilton MG, Lee JS, Cummings PJ, Zabramski JM. A comparison of intra-arterial and intravenous tissue-type plasminogen activator on autologous arterial emboli in the cerebral circulation of rabbits. Stroke. 1994;25:651-656.
Nanda N, Schlief R. Advances in Echo Imaging Using Contrast Enhancement. Amsterdam, Netherlands: Kluwer Academic Publishers; 1993:97-110.
Majno G, Ames A III, Chiang J, Wright RL. No reflow after cerebral ischemia. Lancet. 1967;Sept 9:569-570.
del Zoppo GJ, Poeck K, Pessin MS, Wolpert SM, Furlan AJ, Ferbert A, Alberts MJ, Zivin JA, Wechsler L, Busse O, Greenlee R Jr, Brass L, Mohr JP, Feldmann E, Hacke W, Kase CS, Biller J, Gress D, Otis SM. Recombinant tissue plasminogen activator in acute thrombotic and embolic stroke. Ann Neurol. 1992;32:78-86.
Haley EC Jr, Brott TG, Sheppard GL, Barsan W, Broderick J, Marler JR, Kongable GL, Spilker J, Massey S, Hansen CA, Torner JC. Pilot randomized trial of tissue plasminogen activator in acute ischemic stroke. Stroke. 1993;24:1000-1004.
Huber P. Cerebral Angiography. Stuttgart, Germany/New York, NY: Georg Thieme Verlag; 1982:243-247.
Matsuoka O. Extrapolation From Experimental Animal to Human: Introduction to Comparative Animal Science. Tokyo, Japan: Soft Science Inc; 1980:132-134.
Keller MW, Segal SS, Kaul S, Duling B. The behavior of sonicated albumin microbubbles within the microcirculation: a basis for their use during myocardial contrast echocardiography. Circ Res. 1989;65:458-467.