Horseradish Peroxidase as a Histological Indicator of Mechanisms of Porcine Retinal Vascular Damage and Protection With Perfluorocarbons After Massive Air Embolism
Background and Purpose This laboratory has previously shown that a second-generation perfluorocarbon emulsion (PFE) reduces the severity of cerebral injury induced by air embolism during cardiopulmonary bypass (CPB). Horseradish peroxidase examines vascular permeability and was used in this study of the mechanisms of cellular protection afforded by the PFE.
Methods Twenty domestic pigs underwent CPB with a prime of standard crystalloid or PFE (5 mg/kg) and crystalloid. After 10 minutes on CPB, a 5-mL/kg bolus of room air or saline (control) was delivered via the right carotid artery. The air insult was delivered as either a single bolus or double bolus. After 1 hour of CPB and 1 hour of spontaneous reperfusion, horseradish peroxidase was injected intravenously and circulated for 15 minutes. After euthanasia, both eyes were removed, and the retinas were isolated for histological analysis.
Results Total length of retinal vessels exhibiting horseradish peroxidase extravasation was significantly less in PFE pigs (P<.05). Vascular spasm and red blood cell hemorrhages were unaffected by PFE. PFE pigs exhibited mild to moderate vascular nonperfusion and red blood cell sludging; crystalloid groups demonstrated severe nonperfusion and sludging.
Conclusions Histological staining with horseradish peroxidase indicated that mechanisms of cerebral air embolism include vascular endothelial leakage, vascular nonperfusion, and red blood cell sludging and hemorrhage. Pretreatment with PFE prevented some sequelae associated with massive air embolism and CPB.
More than 300 000 patients in the United States undergo surgery requiring CPB every year. Postoperative stroke occurs with significant incidence (0.9% to 8%) among these patients1 2 3 and is thought to be caused by cerebral embolism (air, microparticulates, or atherosclerotic debris).1 2 Neuropsychological alterations after CPB occur more frequently than strokes and include memory loss and learning, attention, and concentration deficits.1 Twenty percent to 60% of patients exhibit such abnormalities 7 days after bypass, and 30% of these affected patients will demonstrate a permanent cognitive abnormality.2 3 4 Neuropsychological dysfunction can be attributed to microembolism (air or particulate) or cellular injury secondary to ischemia.1 2
PFEs have been investigated as a potential means of cerebral protection when added to the CPB prime. The properties of PFEs that make them uniquely appropriate for use in CPB include enhanced solubility of gases (O2, N2, and CO2)5 and small particle sizes (0.1 to 0.6 μm diameter) compared with RBCs (7 to 8 μm). This reduced size offers an increased particle surface area for gas exchange and improved oxygen diffusion compared with plasma.5 6 In addition, PFEs may have anti-inflammatory properties, which are poorly understood at this time.7 8
These unique properties of PFEs have led us to investigate the potential protective effects of the emulsion on the brain by investigating retinal vascular changes after massive air embolism. The retinal vasculature is an excellent “window to the brain” for several reasons. First, the endothelial cells that separate the retinal tissue from the ocular circulation (the blood-retinal barrier) are comparable structurally and functionally to the blood-brain barrier (endothelial cells that impart permeability of the central nervous system) because retinal tissue is an embryonic outgrowth of neural tube development. For this reason, parallels may be drawn between the two microcirculations.9 10 Second, the ophthalmic artery supplying the retina is the first branch from the carotid artery that delivers blood to the brain; therefore, the retina and the brain should experience similar embolic loads. Most importantly, the retinal microvasculature is available for observation in living specimens and has been investigated with angiography techniques in other studies.11 The retinal microvasculature readily demonstrates embolic load changes during and after CPB. Based on these correlations, retinal cellular damage observed with HRP staining may be used to assess the relative extent of cerebral damage caused by air embolism and the extent of protection afforded by PFE.
The following study attempted to further investigate the efficacy of a 40% vol/vol second-generation PFE in cerebral protection during CPB. HRP staining was used in an attempt to identify histological indications of retinal vascular damage after air embolism and to elucidate both the mechanism of injury from air embolism and the potential mechanism of protection afforded by the PFE.
Materials and Methods
The experiments were performed in strict accordance with the Guide for Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Research Council (National Academy of Sciences, revised 1996) and were approved by the University of Washington Institutional Animal Care and Use Committee.
Twenty domestic pigs (age, 2 to 4 months; mean weight, 20.5 kg) were sedated with an intramuscular mixture of ketamine (22 mg/kg), acepromazine (1.1 mg/kg), and atropine (0.05 mg/kg). Anesthesia was induced by mask inhalation of halothane in 100% oxygen followed by tracheotomy and endotracheal intubation. Anesthesia was maintained with halothane in 100% oxygen. Muscle relaxation during thoracotomy was achieved with pancuronium bromide (1 mg IV).
All animals were monitored with continuous electrocardiography from peripheral limb leads. Transfemoral polyethylene catheters positioned at the midabdominal level were used for fluid administration and arterial sampling. Electroencephalographic tracings were monitored continuously. Rectal temperature readings were used to maintain normothermia (37±5°C). Fluid maintenance was controlled with lactated Ringer’s (4 mL/kg per hour), and arterial pH was maintained with sodium bicarbonate. Phenylephrine or calcium chloride was administered as needed to support arterial pressure during the early reperfusion period. Dexamethasone (4 mg/kg IV) was administered before sternotomy to prevent a species-specific complement reaction to the PFE.
With the pig supine, a median sternotomy was performed, and the right internal carotid artery was isolated. For thoracic access, a partial right anterolateral thoracotomy was also performed in the left lateral decubitus position. In preparation for CPB, the heart and great arteries were isolated; purse string sutures were placed in the right atrium and the ascending aorta. After anticoagulation (heparin sulfate, 300 IU/kg), the right internal carotid artery was catheterized and secured. This carotid line, meticulously flushed of air bubbles, was used for the injection of saline or air during CPB. The ascending aorta was cannulated with a 5-mm arterial cannula, and the right atrium was cannulated with a 28F two-staged cannula. The tip of the venous cannula was advanced to the inferior vena cava. After the cannulae were connected to the CPB circuit, normothermic bypass was initiated.
The CPB circuit was composed of a roller pump (Sorin Biomedical), a soft-shell venous reservoir (Sarns 3M), a hollow-fiber membrane oxygenator (Medtronic Cardiopulmonary), and polyvinyl chloride tubing (Medtronic). A perfusion flow rate of 80 mL/kg was maintained, and the animal was kept at normothermia (37±5°C) for the duration of bypass. The CPB prime used in this study was PlasmaLyte-A (Baxter Healthcare Corp), a crystalloid solution. The PFE used was Oxyfluor (HemaGen). Swine in the crystalloid groups received a prime consisting only of PlasmaLyte-A, while those in the PFE groups received 5 mL/kg of Oxyfluor added to the crystalloid solution before initiation of bypass. Total prime volume in all cases was 1200 mL.
The 20 swine were randomized into one of six experimental groups to determine prime and insult. Ten animals received the crystalloid solution prime: 4 animals were challenged with a single air insult, an additional 4 received a double air insult, and 2 control animals experienced an isovolemic saline insult. Ten additional animals received the PFE prime; the number of animals receiving each type of insult remained the same.
Two different air insults were used in this study. Animals in the single air bolus (SAB) group received 5 mL/kg of air over a 30-second period. Those in the double air bolus (DAB) group initially received 2.5 mL/kg of air over a 30-second period, then a cross-clamp was placed on the left carotid artery for 15 minutes (to prevent the return of air to the heart). An additional 2.5 mL/kg of air was then delivered over 30 seconds and allowed to flush out of the vessels at its own rate. The total volume of air delivered in each case was 5 mL/kg.
The porcine heart was cannulated and placed on bypass with either a combination prime of crystalloid solution and PFE (5 mg/kg) or only crystalloid solution for a 10-minute stabilization period. Next, in the air insult groups, the air embolism was delivered to the right carotid artery as either a single or double bolus as described. The four control swine received a single 5-mL/kg bolus of saline.
From the time of initial insult, normothermic bypass was continued for 1 hour. The animal was weaned from the bypass pump and allowed to reperfuse spontaneously for 1 hour while remaining under general anesthesia. After reperfusion, an HRP staining preparation (type II, 180 mg/kg in 5 mL buffered saline)12 was injected intravenously and allowed to circulate for 15 minutes. At the end of the experimental period, the animal was killed with concentrated pentobarbital (90 mg/kg). Both left and right eyes were removed after euthanasia and placed in cold buffered saline until dissection and staining.
Methods of Measurement
Retinal vascular damage after massive air embolism was assessed with histological staining of the retinal tissues. After enucleation, the retinas of both eyes were isolated and fixed in 50% Karnovsky’s solution12 for 90 minutes. The incubation solution of 250 mL 0.05 mol/L Tris buffer (Trizma base, Sigma), 2.5 mL 3% hydrogen peroxide (York Pharmacal), and 2 mL buffered diaminobenzidine (Sigma) was prepared immediately before use.12 Each retina was incubated for 9 hours in foil-wrapped jars in a dark cabinet to minimize light artifacts.
HRP has been extensively used to study microvascular permeability in the cerebral cortex and other tissues and to study vessel patency and vascular permeability in retinal tissues of diabetic patients.13 The molecular size of HRP (diameter, 2.5 to 3.0 nm) is such that the molecules cannot pass through retinal endothelial junctions (approximately 0.8 nm)9 unless these junctions have been compromised or the functional diameter increased after injury. HRP can be injected directly into the systemic circulation, and results from such studies are highly consistent.12 In addition, HRP produces complete filling of the vascular tree because it is injected before euthanasia; dyes injected postmortem usually produce incomplete vascular filling and inconsistent staining results.12
The retinal tissues were evaluated in a blinded fashion with light microscopy and video microscopy with the use of a 486DX-33 computer (Dell Corp) and image analysis system (Optimas Corp) that produced a ×380 magnification of the retinal vasculature for measurements. The quantified parameters included total length of vessel demonstrating extravasation (“leakage”) of HRP, total length of vessel in spasm, and the number of RBC hemorrhages (or abnormal clusters of RBCs outside of vessels) in each retina.
The severity of vascular nonperfusion and RBC sludging (low flow), as well as vessel spasm severity, was subjectively graded in each tissue according to a scale (mild, moderate, or severe nonperfusion or spasm) established during a pilot study. Vascular nonperfusion was classified according to the degree to which the vasculature was filled with the brown reaction product of HRP. Mild nonperfusion was classified as nearly complete filling of the retinal vascular tree (Fig 1⇓). Moderate nonperfusion was indicated by the presence of vasculature with scattered HRP reaction product within the lumen, and severe nonperfusion exhibited vessels with essentially no visible HRP reaction product within the lumen.
The length of vasculature exhibiting spasm was determined by measuring the total length of all segments of vasculature within a retinal wholemount that appeared to be in spasm. Because it was rare to discover a length of vasculature that showed only one focal spasm, vascular segments showing multiple constrictions (Fig 2⇓) were considered a single data point. The total length of the constricted segment was determined and added to the other lengths of vasculature to determine the total length of vasculature in spasm evaluated in that retinal wholemount.
Results were analyzed for statistical significance with factorial ANOVA and unpaired t tests (StatView, Abacus Concepts, Inc), with P<.05 considered significant. We performed 2×3 factorial ANOVA to find significance for the effect of prime (PFE versus crystalloid) or insult (saline, SAB, or DAB). Unpaired t tests were performed for comparison of prime effect within a given insult group. Data are presented as mean±SEM.
Vascular HRP Leakage
Fig 1⇑ is representative of retinal vasculature exhibiting HRP extravasation after air embolism. Regardless of type of insult (single or double bolus), there was significantly less total length of leaking retinal vessel in PFE-primed animals than in crystalloid-primed animals (P<.05 ANOVA). When primes were compared (PFE versus crystalloid) for a given insult type (unpaired t test; Table 1⇓), the saline insult group showed no significant difference (P=.90) in length of vascular leakage, although a small amount of HRP extravasation was observed in these animals. The SAB insult group demonstrated a trend for the PFE group to have less HRP leakage than the crystalloid group (P=.15), and the DAB insult group showed significantly less HRP leakage in the PFE group than the crystalloid group (P<.05).
An example of vascular spasm in retinal tissue from this study is shown in Fig 2⇑. There was no significant difference in total length of vessel in spasm in PFE-primed animals than in crystalloid-primed animals when evaluated for effect of prime (P=.30) and insult (P=.16). When primes were compared for a given insult (Table 2⇓), the saline insult groups showed no significant difference (P=.67) for length of vessel in spasm. The SAB insult groups (P=.25) and the DAB insult groups (P=.88) also showed no significant differences between primes.
Fig 3⇓ shows an example of a localized RBC hemorrhage in retinal tissue. There were no significant differences in number of RBC hemorrhages between groups when evaluated for effect of prime (P=.67). There was, however, a significant difference for the effect of insult (P<.01). The SAB groups showed significantly more RBC hemorrhages than the saline groups (P<.005), as did the DAB groups compared with saline groups (P<.01), regardless of receiving a PFE or crystalloid prime. There was no significant difference in the number of RBC hemorrhages between the two different types of air insult (SAB versus DAB; Table 3⇓).
Subjective Assessment of Nonperfusion and Vessel Spasm Severity
All PFE-primed pigs and crystalloid-primed/saline-insult pigs demonstrated mild to moderate nonperfusion; there were no examples of severe nonperfusion. In contrast, crystalloid-primed pigs that received the air insults demonstrated some moderate but primarily severe nonperfusion.
The severity of retinal vascular spasm ranged from mild to severe in all groups; there was no apparent correlation with prime or insult. Vascular spasms also did not correlate with areas of nonperfusion.
Unexpected Observations: Microvascular Inclusions in Retinal Vessels
Upon inspection of the HRP wholemounts at ×125 magnification, small inclusions were noted in the vessels of those animals receiving the perfluorocarbon prime. Benchtop tests failed to discern the makeup of the vascular inclusions noted in these animals. It was hoped that an established lipids test (Oil Red O) would demonstrate that the emulsion had “broken” or separated into its constitutive elements of PFC and lipid emulsifier to cause the bubble-like inclusions postmortem. However, it was unclear what a negative Oil Red O test would be when used in the presence of a PFE. These inclusions were opaque and nonstaining and often appeared to cause distention of the vessels. The average diameter of these inclusions was measured to be 13.8 μm with the use of the Optimas imaging system. Further inspection showed the inclusions to be exclusively associated with the PFE animals. However, it was noted that these inclusions were not associated with any of the aforementioned indicators of vascular damage; no areas of vascular leakage, RBC hemorrhages, or spasm were noted. The presence of RBCs was also noted on both distal and proximal sides of the vascular inclusions.
In this study HRP histological staining demonstrated significantly less HRP leakage in the PFE-primed pigs than the crystalloid-primed pigs. This result suggests that PFE does protect the endothelial cells of the retinal vessels from changes that would allow the extravasation of HRP. Since HRP is a relatively large molecule (2.5 to 3.0 nm in diameter), the effective diameter of the endothelial junctions would have to increase three to four times normal size to allow dye particle extravasation.
The total lengths of leakage noted in each animal group depended not only on the prime but were also representative of the severity of the air insult. For example, the PFE pigs all demonstrated comparable lengths of vascular leakage (2627 to 3858 μm), as did the crystalloid/saline groups. The crystalloid/SAB group, however, showed a threefold to fourfold increase in total length to 12 477 μm, and the crystalloid/DAB group showed an even greater total length of 14 254 μm. The severity of leakage in the crystalloid/air insult group compared with the PFE/air insult group clearly shows that the PFE minimizes the effects of air embolism on endothelial permeability. The minimal lengths of leaking vessels shown in the PFE group and the crystalloid/saline group may be a “baseline” level of leakage due to the complement activation following the initiation of CPB14 or other inflammatory responses, including white cell activation or reperfusion injury. However, vascular permeability was typically not diffuse but limited to multiple short lengths of vasculature, suggesting that the endothelium was not equally affected, as might be expected if complement activation were the cause. In this case, the baseline levels of leakage noted in the saline bolus pigs may represent lower levels of gaseous or microparticulate embolic injury.
The lack of significant effect of prime or insult on the appearance of vascular spasm indicates that spasm is most likely the result of a factor common to all of the groups. Vascular spasm has been noted to be produced in other studies by the presence of HRP.15 Vasospasm may also be part of the inflammatory response to CPB. Once again, vascular spasm was not universal but occurred in selected vessels. In Table 2⇑, the lower mean value for the PFE/SAB group deserves note, although it was not statistically different from the crystalloid/SAB group (P=.25). The possibility exists that vascular spasm in PFE-primed animals is decreased with reduced air embolism; additional work would be required to provide an answer to this question.
RBC hemorrhages were almost exclusively associated with pigs receiving air insults, possibly indicating that the air insult altered the permeability of the endothelial junctions to the extent that RBCs could escape. RBC hemorrhages were typically even more localized than the HRP leakage, suggesting the injury of only a few endothelial cells that may be the result of direct contact from a bubble lodged in the vessel lumen. In fact, RBC hemorrhages were often noted at points of vessel branching where an obstruction would most likely lodge. In contrast, the HRP leakage may be the result of more diffuse endothelial injury, possibly caused by ischemic injury to endothelial cells downstream of the obstruction. Because RBCs (7 to 8 μm) are larger than HRP molecules (0.0025 to 0.003 μm), the most severe or prolonged injury to the endothelial cells would be required to allow their extravasation.
Nonperfusion or “sludging” of the RBCs within the vessels was a potential indication of rheological change, or low flow, caused by microembolism. In those pigs not primed with PFE, RBCs often appeared collected along the sides of a retinal vessel or were absent in a particular vessel, perhaps indicating reduced blood flow or a vascular occlusion before time of death. The retinal tissues, however, were removed immediately after euthanasia, minimizing the time for sludging to occur from death alone. The reduced sludging in PFE-primed animals indicated that the PFE had substantial beneficial effect in maintaining blood flow in the microcirculation and in preventing the sludging of blood cells.
The unexpected findings of vascular inclusions noted in the PFE animals in the HRP wholemounts are almost certainly related to the PFE. There is no definitive explanation for the origin of these inclusions, but certain observations may help in understanding them further. The inclusions are present in vessels that appear to be venules, and they are not associated with any signs of hemorrhage, vascular leakage, or other signs of tissue damage. RBCs are clearly visible on both sides of the inclusions, indicating that these events did not occlude blood flow before the animal’s euthanasia. There is a suggestion of birefringence of these inclusions, much like intramacrophage PFE. Although we cannot be sure, it is believed that these inclusions represent circulating PFE, possibly altered by the conditions of euthanasia and/or the fixation and staining process.
The mechanisms of cellular damage in the retina due to air embolism and affected by the presence of PFE were represented by vascular leakage of HRP molecules, RBC hemorrhages and sludging, and vascular nonperfusion. Vascular spasm did not appear to be influenced by either air insult or use of PFE as a CPB prime. The results of this study indicate that the PFE Oxyfluor does protect the retina from the effects of a massive air embolism during CPB procedures by limiting vascular endothelial damage and improving perfusion. Although not all results were statistically significant, the trends consistently point to the efficacy of the PFE in cerebral protection.
This study was designed with the use of a massive air embolism to model a “worst-case” scenario; it is reasonable to assume that since the PFE did exhibit potential benefits with this model, it would be as or more effective in the event of air microembolism. In addition to providing protection from air microembolism, the properties of PFE may also be amenable to minimizing the impact of other types of embolic events that lead to neurological dysfunction. PFE may potentially reduce the effects of microparticulate emboli, such as thrombus or atherosclerotic debris, as well as gaseous microemboli. The results of this study regarding vascular perfusion and RBC sludging also indicate that PFE may have a beneficial effect on low- or no-flow states that can lead to hypoxia and neurological damage.
These data support a preservation of endothelial integrity when challenged with a massive air embolism. Cerebral air embolism has previously been shown to trigger a thromboinflammatory response leading to secondary injury.16 17 18 19 The data indicating no difference in major hemorrhages may indicate that the PFE is not rapidly absorbing very large air bubbles, but it is still unclear whether the PFE may be capable of absorbing smaller bubbles. PFE may not be able to absorb the larger bubbles rapidly enough to minimize the occurrence of cerebral damage, since cerebral injury occurs within 4 to 8 minutes. Future work is needed to elucidate whether the endothelial preservation noted in this study is due to enhanced oxygen delivery, increased microbubble dissolution, or endothelial inflammatory preservation of homeostasis. Work in human CPB is necessary to determine whether the protection demonstrated in these laboratory animals can be translated into preservation of human brain function.
Selected Abbreviations and Acronyms
|DAB||=||double air bolus|
|RBC||=||red blood cell|
|SAB||=||single air bolus|
This study was supported in part by a research grant from HemaGen Inc. of St Louis, Mo, as well as departmental institutional research funding. Ms Herren was supported by a Tau Beta Pi Graduate Fellowship and National Science Foundation Graduate Fellowship throughout this study. The authors would also like to acknowledge the assistance of Hiroji Akimoto, MD, Robert Thomas, BA, Christine Rothnie, LVT, Gina Lindley, BS, and the expert pathology assistance of Sidney Goldfischer, MD, regarding the microvascular inclusions.
Reprint requests to Janelle I. Herren, MSE, Baxter Healthcare, Inc, Blood Substitutes Division, WG3-1S, Route 120 and Wilson Rd, Round Lake, IL 60073.
- Received March 31, 1997.
- Revision received June 7, 1997.
- Accepted June 18, 1997.
- Copyright © 1997 by American Heart Association
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