Contrast agents have long been used in the practice of echocardiography to aid in delineation of both the structure and function of the heart. Gramiak and Shah (1) first reported use of agitated saline to improve M-mode imaging of the aortic root. For many years, agitated saline was the only contrast medium available and was used primarily to test for the presence of right-to-left intracardiac shunt, as might be seen in the presence of a patent foramen ovale. This technique has only been effective in the right side of the heart after an IV injection, as the air bubbles do not cross the pulmonary vascular bed.
In the last few years, there has been an effort to develop agents that will cross the pulmonary circulation and allow imaging of the left side of the heart after IV injection. The first of these agents was Albunex™ (Molecular Biosystems, San Diego, CA), a compound composed of 5% sonicated human albumin. Clinical experience with Albunex has been variable (2,3) with some authors reporting the necessity of left-heart injection, as during cardiac surgery, to obtain adequate left-heart images (4). Investigational work is in progress using Albunex as a marker for myocardial perfusion (5), but limitations of its characteristics have led to the development of the second-generation agents, such as Optison.
Optison™ (FS069; Mallinkrodt Inc., St. Louis, MO) is one of a group of “second-generation” transpulmonary echocardiographic contrast agents. These agents are specifically designed for injection into the venous circulation with transpulmonary passage to the left side of the heart (6). Optison is widely used to improve echocardiographic delineation of myocardial structures in adults. Although there are anecdotal reports of its use in children (L. Huffman, Mallinkrodt, Inc., oral communication, 1998), the manufacturer specifically cautions against its use in children and all patients with suspected or known congenital cardiac anomalies. Although Optison would be very useful in the echocardiographic evaluation of complex congenital heart disease, there is concern that if introduced directly into the cerebral circulation, as in the case of a right-to-left shunt, possible deleterious effects could occur from persistence of large gas particles in the cerebral circulation. We examined the effects of Optison on the blood-brain barrier of rats when injected directly into the cerebral circulation.
Methods
After approval by the institutional animal use committee, and in accordance with NIH guidelines, male Sprague-Dawley rats were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and were allowed to breathe spontaneously. Anesthesia was maintained by repeat injections of ketamine and xylazine as needed. Under sterile conditions, the left carotid artery was dissected and cannulated with a PA-50 tube, as were the left femoral vein and artery. Arterial blood pressure was monitored continuously via the left femoral artery only at periods of 90 min and 180 min after Optison injections.
Animals were divided into two experimental groups (n = 15 per group) and received an injection of either 2.5 or 5 mg/kg of Optison into the carotid artery for 60 s by using a Harvard Infusion Pump (Harvard Apparatus Inc., Holliston, MA). After the Optison injection, each experimental group was divided into three subgroups and received Evans blue dye (1 mL/kg, 2%, injected 20 min before the animals were killed) at intervals of 90 min, 180 min, and 24 h via the femoral vein. The uptake of Evans blue dye into the capillary wall served as a marker to indicate disruption of the blood-brain barrier. Two animals received mannitol (1 mL/kg) followed by Evans blue dye 90 min later. Because mannitol is known to transiently disrupt the blood-brain barrier, these animals served as positive controls. A control group (n = 5) received saline (2 mL/kg) followed by an injection of Evans blue dye (1 mL/kg) 90 min later. Animals were not exposed to ultrasound at any time. All animals were killed 20 minutes after the Evans blue dye injection.
Evans Blue Staining
The brains of rats receiving saline injection served as control. The color of the brain was pinkish without blue staining. After fixation in glutaraldehyde as part of the tissue preparation process, the color changed to white. White was regarded as Grade 1. Light bluish staining in some areas of the brain was regarded as Grade 2. Marked staining in multiple areas was regarded as Grade 3. Dark blue over the whole brain was regarded as Grade 4 staining.
Tissue Preparation for Microscopic Studies
Rats were killed with an overdose of ketamine (500 mg/kg) and xylazine (50 mg/kg). Animals were perfused via a cardiac catheter with isotonic saline and 2% glutaraldehyde in 0.1 M Sorenson’s phosphate buffer at a perfusion pressure of 100 mm Hg. Whole brains were removed immediately and placed in 2% glutaraldehyde for 24 h. To preserve Evans blue dye potentially within the cerebral tissue, routine histological staining methods were not used. These unstained brain samples were cut by using a vibratome, which represents an alternative method of sectioning tissue. Cross sections of unstained brain pieces were mounted on the tissue chuck with a cyanoacrylate glue, placed in the bath, and covered with 0.1 M sodium phosphate buffer. Samples were cut at 50 μm, mounted on gelatin-subbed slides, allowed to air dry overnight, and then coverslipped with cytoseal 60 mounting medium. To permit evaluation of the general anatomical integrity of cerebral tissue and related neurons, some pieces of brain from both experimental groups were paraffin embedded, sectioned (8 μm) and stained with H & E (hematoxylin and eosin).
Blood pressure data were analyzed by using analysis of variance with P < 0.05 considered statistically significant. The Evans blue staining data were analyzed by using the unpaired student’s t-test.
Results
Blood Pressure Changes
Figure 1 summarizes blood pressure changes for a period of 90 and 180 min after the Optison injection. After some transient but insignificant increases (P > 0.05, analysis of variance) immediately after the Optison injection, blood pressures had returned to normal ranges by 30 min. Likewise, at 90 and 180 min after the Optison injection, blood pressures were within normal ranges. There were no significant differences between the blood pressure before or after the Optison injection.
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Figure 1: Blood pressure values before, during, and after the Optison injection. By 90 min (A) and 180 min (B), blood pressures were within normal range. The transient increase of blood pressure during the Optison injection was not significantly different from the values collected at other times (P > 0.05, analysis of variance).
Gross Examination of Brains
Figure 2 demonstrates the appearances of gross brains for experimental and control animals. After Optison (at both dosage levels) and Evans blue dye injections (at all three time periods) the surface of gross brains demonstrated varying degrees of blue staining that appeared to be Optison-dosage-dependent. Figure 2A demonstrates the typical appearing gross brain 90 min after a 2.5-mg/kg Optison injection. The brain surface demonstrated only a light bluish tint. Figure 2B is a typical brain of a 5-mg/kg Optison injection at 90 min. The entire brain surface showed intense blue staining. Figure 2C illustrates the brain of a saline injected rat at 90 min. The surface of the control brain did not show any blue tint and had a whitish color. The staining of gross brain tissue in this study demonstrated an Optison-dosage-dependent effect. Mannitol-(1 mL/kg) injected rats, as anticipated, demonstrated dark blue staining of the brain surface (not shown).
Figure 2: A, Brain of a 2.5 mg/kg Optison-injected rat at 90 min. The brain surface demonstrates only a light bluish tint. Some areas stained darker (arrows). B, Brain of a 5 mg/kg Optison-injected rat at 90 min. Note that entire brain surface is stained dark blue. C, Brain of saline-injected rat at 90 min. The surface of brain is unstained, and its coloration is normal.
To further quantify the gross examination of the Evans blue dye staining, each brain was scored on a scale of 1 to 4 as described in the Methods section. For example, the samples shown in Figure 2; A and B, were scored as 2 and 4, respectively. Figure 3 demonstrate a concentration-dependent effect of Optison on grade of staining at 90 min (Figure 3A) and 180 min (Figure 3B), respectively.
Figure 3: Summary of scale of Evans blue dye staining is rats at 90 min (A) and 180 min (B). The larger dose of Optison (5 mg/kg) produced more and darker staining than the smaller dose (2.5 mg/kg) in the brains. *P < 0.05 (unpaired student’s t-test).
Vessel Histology
Light microscopic examination of brain sections revealed that Evans blue dye uptake into the cerebral capillary wall was Optison-dosage-dependent. In rats that received 2.5 mg/kg Optison at various time intervals, dye uptake into the capillary walls could only be vaguely identified (not shown). However, in rats that received 5 mg/kg Optison, an extensive capillary network was clearly visible marked by the uptake of Evans blue dye at all time intervals. The capillary walls of saline injected animals showed no evidence of Evans blue staining. Figure 4 shows examples from three rats that were killed at 90 min, 180 min, and 24 h after 5 mL/kg Optison injection (Evans blue dye was injected 20 min before death). Apparently, the blood-brain barrier was disrupted even 24 h after Optison injection. Even though blood-brain barrier disruption continued at 24 h, no neuronal damages could be revealed by light microscopy at any time period. H & E sections of brains revealed normal cerebral architecture with a rich abundance of glial cells and neurons (Figure 5). Although no Evans blue dye was observed, it could have been dissolved by the alcohol in the H & E staining method. There was no evidence of an inflammatory response in any section.
Figure 4: Cerebral capillaries of a 5 mg/kg Optison-injected rat at 90 min (A), 180 min (B), and 24 h (C). Arrows and arrowheads illustrate dark staining Evans blue dye within capillary walls.
Figure 5: Hematoxylin and eosin section of a 5 mg/kg Optison-injected rat at 90 min. The cortical tissue appears normal with no visible evidence of Evans blue dye. Arrows indicate pyramidal cells.
Discussion
Optison consists of octofluoropropane-filled human albumin microspheres with a mean bubble size of 3.6 μm and mean concentration of 8 × 108 mL (7). Octofluoropropane gas (C3F8) is used commercially as a cleaning medium for chemical vapor deposition vessels and in processes such as the etching of electronic circuit boards. It has a molecular weight of 188 daltons with a boiling point of −37°C, freezing point of −183°C, and vapor pressure of 114.8 psia (at 21.1°C) (8). Plasma energy breaks octofluoropropane into active fluorine species, which etch and remove dielectric film build-up. In medical applications, octofluoropropane is generally thought to be inert and rapidly eliminated by the lungs. Because of the strength of carbon-fluoride bonds in biologic systems, it has been assumed that octofluoropropane has no effects on tissue, although it has been noted to have a high tissue affinity (9). Optison injected into a peripheral vein rapidly opacifies the right heart and clearly delineates structures, appearing much like a ventriculogram during cardiac catheterization. Shortly thereafter, the left heart is opacified, followed by a gradual intensification of the echogenicity of the myocardium itself, reflecting myocardial perfusion.
These superb imaging characteristics offered by Optison have caused it to rapidly achieve widespread use. One exciting potential of contrast echocardiography, and specifically use of Optison, is the evaluation of congenital heart disease. Frequently, children have complex congenital heart malformations diagnosed by using current methods of echocardiography, without catheterization. These patients often present for cardiac surgery with therapeutic plans made based on these echocardiograms. Often, additional or different lesions are discovered at the time of surgery. More importantly, echocardiography is often relied on immediately after repair to assist in weaning from cardiopulmonary bypass and determining the adequacy of the surgical reconstruction. A second-generation agent, such as Optison, would be invaluable in better assessing the heart’s structure after repair and evaluating function, as in the case of residual shunts. Unfortunately, the manufacturer cautions that Optison not be used in children or any patients with congenital heart anomalies because of the fear of deleterious cerebral effects if it does not cross the pulmonary circulation before entering the left heart. Despite this, we are aware of Optison use in such children without ill effects.
This study sought to determine whether Optison had an effect on the blood-brain barrier of rats when injected directly into the cerebral circulation. Dosages used varied between 10 and 1000 times the recommended human mL/kg dose, the variability dependent on whether the drug indication was to delineate borders, enhance cardiac structures, or demonstrate myocardial perfusion. Based on body surface area, however, the dosages used were closer to the human dose and similar to those used by the manufacturer in toxicity studies. The Optison was injected into the carotid artery and integrity of the blood-brain barrier was tested at various times after injection by the administration of Evans blue dye. Evans blue dye will be trapped in the vessel walls of cerebral capillaries when the blood-brain barrier has been disrupted, causing them to take on a characteristic outlined appearance (Figure 4). Where the blood-brain barrier is intact, the Evans blue dye will simply pass through the circulation without staining the capillaries. Additionally, the brain will be grossly stained blue when the blood-brain barrier has been disrupted (Figure 2).
In this study, Optison at all dosages disrupted the blood-brain barrier for up to 24 hours. It is likely that this disruption persists even longer, but this extended time interval was not studied. The mechanism of this duration of disruption is unclear. Mannitol, which served as a positive control, disrupts the blood-brain barrier, but only for a limited time. Staining by Evans blue dye after mannitol administration will generally not persist beyond 120 minutes. Mannitol functions as a model for disruption of the blood-brain barrier by a hyperosmolar fluid. Optison, however, is isoosmolar. An increase in blood pressure will also transiently disrupt the blood-brain barrier, but we observed only mild and self-limited brief increases in blood pressure after injection. Although an inflammatory response of the rat to human albumin is a possibility, histologic examination of the cerebral tissue revealed no visible evidence of an inflammatory response. This leads us to believe that the damage sustained by the blood-brain barrier of the rats was a result of a tissue effect of the octafluoropropane gas itself.
Optison has not been shown to have adverse hemodynamic, hematologic, or inflammatory effects in animal or human studies at therapeutic dosages (10,11). Detailed studies of its effects on the central nervous system have not been done. Cell lysis and sonoporation by ultrasound is enhanced by the use of contrast agents (12). Optison, in particular, increases cell destruction by ultrasound as compared with Albunex (13). This is thought to be caused by both whole gas bubble effects on tissue, as well as cavitation nuclei of disrupted bubbles. Additionally, ultrasonic contrast agents generate significant subharmonics when insonated and may further contribute to tissue damage (13). Miller and Gies (14) have shown that Optison causes greater degrees of hemolysis on ultrasound exposure than the other second-generation agents, and far more than Albunex. The qualities of Optison that cause it to persist and enhance its ability as a contrast agent may account for this difference. It is important to again note, however, that the animals in our study were not exposed to ultrasound. It is possible that a localized large concentration of Optison, even in the absence of ultrasound exposure, as in our study, may have the same effect on cell membranes as smaller concentrations of Optison exposed to ultrasound energy; however, further studies are necessary to address this.
Finally, this study did not address the pathologic consequence to the disruption of the blood-brain barrier. Although we noted no gross neurologic abnormalities in the animals up to 24 hours after injection, this study did not specifically test for neurologic change, nor did it take into account effects of the initial anesthetic. In addition, no neuronal damage was revealed in the presence of disruption of blood-brain barrier (Figure 5).
In conclusion, Optison, when injected directly into the cerebral circulation of rats, disrupts the blood-brain barrier. This disruption is persistent and, in this study, lasted at least 24 hours. The dosages we used were significantly larger than equivalent human doses, and further study is required to address the effects of Optison when used in clinical doses. The disruption of the blood-brain barrier may be a direct effect of octofluoropropane gas on cell membranes. Other studies have demonstrated that Optison enhances tissue damage by ultrasound energy (12,14), and it is possible that the large doses used in this study, even without ultrasound exposure, mimic this phenomenon. Further investigation of the neurologic safety of Optison in the cerebral circulation is necessary, because it could be significantly useful in the evaluation of complex congenital heart disease.
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