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Ann Thorac Surg 1996;62:1313-1320
© 1996 The Society of Thoracic Surgeons
Division of Cardiothoracic Surgery and the Kennedy Krieger Institute, Johns Hopkins Medical Institutions, Baltimore, Maryland
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Abstract |
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 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Methods. Adult male hound dogs (n = 17) were subjected to 2 hours of HCA at a brain temperature of 18°C and reperfused to normothermia; they were sacrificed at various intervals up to 74 hours. Using in vivo cerebral microdialysis, dogs (n = 5) were given a simultaneous infusion of artificial cerebrospinal fluid containing L-[14C]arginine or L-[14C]arginine and L-nitroarginine methyl ester (a nitric oxide synthase inhibitor) in contralateral hemispheres while undergoing 2 hours of HCA and reperfusion to normothermia.
Results. L-[14C]citrulline recovery, a coproduct of nitric oxide, significantly increased during HCA in the hemisphere without the inhibitor (at 300 minutes: control, 236 ± 94 fmol/min versus L-nitroarginine methyl ester, 6 ± 6 fmol/min; p < 0.05). Citrulline production in vitro from canine cortical homogenates in the presence of calcium (n = 12) was significantly greater 8 and 20 hours after reperfusion (5.11 ± 0.54 x 10-7 mmol • mg-1• min-1 and 7.52 ± 0.59 x 10-7 mmol • mg-1 • min-1, respectively) than before HCA (1.51 ± 0.09 x 10-7 mmol • mg-1 • min-1; p < 0.05). Nitric oxide metabolites in the serum were also increased significantly early after reperfusion (baseline, 6.72 ± 0.95 mmol/L; at 4 hours, 17.58 ± 1.46 mmol/L; p < 0.05). Immunocytochemical staining of the cortex with neuronal nitric oxide synthase-specific monoclonal antibodies (Transduction Labs) revealed increased neuronal nitric oxide synthase expression 6 to 18 hours after HCA. Darkfield analysis demonstrated neuronal nitric oxide synthase localization to neuronal processes with widespread formation of dense plexi of nitric oxide synthase fibers.
Conclusions. We conclude that neurotoxicity after HCA involves a significant, early induction in neuronal nitric oxide synthase expression in neuronal processes leading to widespread augmented nitric oxide production in the brain.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Hypothermic circulatory arrest (HCA) has become the surgical technique of choice for complex operations involving the heart and great vessels. Experimental and clinical evidence has validated this technique of HCA during which an arrested patient can be maintained without cardiopulmonary support. However, neurologic outcomes after HCA have been found to be directly related to the length of circulatory arrest. Arrest times exceeding the widely practiced "safe" time of 60 minutes have been associated with stroke and cognitive deficits.
The exact mechanism or cascade of events leading to neuronal death is unclear. Understanding this mechanism may provide an additional means to inhibit neurologic injury. There is some evidence that abnormally high concentrations of nitric oxide (NO) in the brain may exert neurotoxic effects. We hypothesized that excessive extra-cellular accumulation of NO in the brain may be associated with HCA-induced cerebral injury.
A series of experiments was designed to assess NO production during HCA. These included in vitro assays of NO synthase (NOS) activity in cerebral tissue homogenates, in vitro assays of NO metabolites in serum, a qualitative immunohistochemical study of the canine cerebral cortex, and a method of in vivo detection of NO production in the brain that we recently developed using microdialysis probes [1].
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Bilateral tympanic membrane probes (which correlate closely with cerebral temperature) [5], nasopharyngeal temperature probes, and rectal temperature probes were placed. Monitoring of blood pressure and arterial blood gas determinations were made via a left femoral artery catheter. Standard electrocardiographic monitoring was employed. Arterial blood gas management was accomplished using the alpha-stat technique. Animals were prepared and draped sterilely. A left external jugular vein catheter was placed percutaneously for administration of fluids and drugs.
Cardiopulmonary Bypass and Hypothermic Circulatory Arrest
Cardiopulmonary bypass (CPB) was conducted using a Bentley-10 Plus bubble oxygenator (Baxter Healthcare, Irvine, CA), a Sarns roller pump (Sarns Inc, Ann Arbor, MI) and a 40-µm in-line arterial filter. The circuit prime consisted of 1 L of lactated Ringer's solution with 20 mEq of sodium bicarbonate and 10 mEq of potassium chloride. After systemic heparinization (300 U/kg), venous cannulas (16 to 18F) were advanced to the level of the right atrium through both the right femoral and right external jugular veins. The arterial cannula (12 to 14F) was placed in the descending aorta via the femoral artery. Cardiopulmonary bypass (CPB) was then initiated and the animal cooled to 18°C (tympanic membrane temperature) with core (CPB) and surface cooling (ice bags around the head and cooling blanket). During CPB, mean arterial blood pressure was maintained at 50 to 55 mm Hg with pump flows of 80 to 100 mL/kg and reduced to 50 to 60 mL/kg when the tympanic membrane temperature was less than 32°C. A tympanic membrane temperature of 18°C was achieved within 25 to 30 minutes of the initiation of CPB. At this point, the arterial pump was turned off, and the venous blood was drained by gravity into a cardiotomy reservoir.
Circulatory arrest was maintained for 2 hours (to ensure neurologic injury in all animals) at 18°C followed by the reinstitution of CPB and rewarming. Dogs received sodium bicarbonate to maintain a base deficit less than 7 mEq. At normothermia, animals were weaned from CPB support and decannulated, and heparin was reversed with protamine. The right femoral artery and vein were then ligated and the wounds closed in layers. The excellent collateral flow of the dog's lower extremities prevented ischemic neuropathy of the distal aspect of the limbs.
Postoperatively, animals remained on the ventilator and underwent intensive care monitoring to ensure strict respiratory and hemodynamic control. Dogs were maintained on halothane (1.0%) and fentanyl as needed (20 µg/kg intravenously) and arterial blood gases, hemoglobin, glucose, cardiac rhythm, arterial blood pressure, and urinary output were closely monitored with appropriate interventions as necessary.
At 8, 20, and 74 hours after reperfusion following HCA, 4 animals were sacrificed fully anesthetized at each time period. The 4 animals that survived for 74 hours, were awakened after 20 hours, weaned from ventilatory support, and extubated. Neurologic assessments were performed every 12 hours, and postoperative pain control was administered with morphine sulfate, 1 to 3 mg every 2 to 3 hours intravenously for the first 24 hours. Our neurologic assessments in these dogs and their outcomes have been described in detail previously [2–4], and were performed here to ensure the reproducibility of severe brain injury in this model. The 4 animals that survived for 3 days and underwent neurologic assessments all displayed significant cognitive impairment consistent with previous reports of this standard model. Seventy-four hours after reperfusion, the animals were reanesthetized, reintubated, and ventilated. A median sternotomy was performed, followed by clamping of the ascending and descending aorta. While fully anesthetized, dogs were sacrificed by exsanguination (consistent with American Veterinary Medical Association recommendations) with perfusion of the brain for fixation performed at 100 mm Hg pressure via the aortic cannula. The brains were then harvested and cut into 1-cm blocks. Five control dogs, which did not undergo HCA, were sacrificed by exsanguination while fully anesthetized, and their brains were harvested in a similar fashion to the dogs in the experimental group.
Intracerebral Microdialysis
Microdialysis probes were constructed as described by Johnson and Justice [6] and Van Wylen and associates [7]. Two silica perfusion tubes (inner diameter, 75 µm; outer diameter, 150 µm; SGE, Austin, TX) were sealed in a segment of hollow dialysis tubing (inner diameter, 300 µm; membrane molecular weight cutoff, 5,000 Daltons) so that their open ends were 10 mm apart. These open ends serve as inflow and outflow cannulas, whereas the distal tips are sealed with epoxy. The effective dialyzing area of the membrane is the 10-mm distance between the inflow and outflow cannulas. The skull of each animal was exposed bilaterally and the coronal suture identified. Burr holes were placed bilaterally 8 mm from the midsagittal axis and 3 mm caudally from the coronal suture. Microdialysis probes were placed to a depth of 20 mm stereotactically into the corpus striatum using a micrometer. Probes were fixed with dental cement. The tissue was allowed to stabilize from the trauma of probe placement for 60 to 90 minutes. Warmed artificial cerebrospinal fluid (CSF) (mmol/L concentration; NaCl, 131.8; NaHCO3, 24.6; CaCl2 2.0; KCl, 3.0; MgCl2, 0.65; urea, 6.7; and dextrose, 3.7) was filtered and continuously bubbled with 95% N2 and 5% CO2 along with 3 µmol/L [14C]-L-arginine (DuPont-New England Nuclear) until O2 and CO2 tensions were similar to those of CSF and brain tissue. The CSF and arginine then were infused at 2 µL/min through the dialysis cannula and collected by drip from the outflow cannula into preweighed plastic vials. In the contralateral hemisphere, 1 mmol/L L-nitroarginine methyl ester (L-NAME) was added to the radioactive CSF and infused similarly at 2 µL/min. Serial collections of 60-µL effluent dialysate samples from each hemisphere were obtained every 30 minutes, stored at -85°C, and then assayed for citrulline activity as a marker of NO production. Samples were diluted with 200 µL of water and poured over Dowex-50W (Sigma #50X8-400) columns which bind charged arginine but allow the passage of neutral citrulline. Columns were then washed with 2 mL of buffer containing 30 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) (pH 5.2), 3 mmol/L EDTA, and 1 mL of water. In addition, 60 µL of radioactive CSF, not used for dialysis, was diluted with water and poured over a Dowex column to determine the resin efficiency of arginine trapping. The radioactivity of the effluent was quantified using a liquid scintillation counter. Background activity was determined by the column flow of the nondialyzed artificial CSF samples. Specific activity was corrected for counting efficiency and background activity, and was expressed in femtomoles per minute of perfusion. Finally, the [14C]-L-arginine content of 100 µL of artificial CSF not used for dialysis was measured as a control to ensure that consistent concentrations of radiolabeled arginine were used.
Nitric Oxide Synthase Assay
Animal tissues were collected and homogenized in a volume of sample buffer 10 x sample weight. Sample buffer was 50 mmol/L Tris, pH 7.4, 1 mmol/L EDTA, 1 mmol/L EGTA. The homogenate was mixed with 1 mmol/L CaCl2 and 10 mmol/L nicotinamide adenine dinucleotide phosphate (reduced form) and 3H-arginine (100,000 cpm) for a total of 200 mL, and incubated at 22°C for 15 minutes. The reaction mixture was diluted with 3 mL 20 mmol/L Na-free HEPES and 2 mmol/L EDTA and poured over a column of Dowex-50W. The effluent was counted in a scintillation counter. This represents the amount of arginine converted into citrulline in vitro. The advantage of this system is that it is sensitive and rapid.
Nitric Oxide Breakdown Product Assay: The Griess Reaction
The Griess reaction was first developed to measure nitrite, and has subsequently been used to measure the products formed from NO in vivo. The Griess reaction measures only NO2-, but NO forms both NO2- and NO3-, so to measure the total NO, the NO3- in the sample must first be converted back into NO2-. Thus a measurement of NO2- plus NO3- in animal fluid is the most reliable assay for NO production.
Serial samples of blood were collected every 2 hours for the first 20 hours, centrifuged to remove excess protein, stored at -85°C, and analyzed for nitrate and nitrite using the Griess reaction. The NO3- is converted to NO2- by enzymatic reduction of nitrate to nitrite by Aspergillus nitrate reductase (Boehringer Mannheim) at 37°C for 30 minutes. An equal volume of sample was then added to the Griess reagent (50 mL of 1% sulfanilamide, 2.5% H3PO4, and 50 mL of 0.1% napthylethylenediamine) and incubated for 30 minutes at 22°C. A set of NO2- standards was also measured to calibrate the readings. The optical density of the sample was read at 546 nm.
Immunocytochemical Staining
Immunocytochemistry uses antibodies to localize antigens on tissue slices. The brain is harvested, blocked, prefixed in 4% paraformaldehyde for 4 to 6 hours, then cryoprotected in 30% sucrose in phosphate-buffered saline solution until the brain sinks. Sections are preincubated for 1 hour in a solution containing 2% NGS, 0.2% gelatin and 0.3% Triton. Sections are incubated (for 48 to 74 hours at 4°C) in primary antiserum (1:200 for NOS antibodies purchased from Transduction Labs) in the same solution. The antigen-antibody complex is visualized using the avidin-biotin peroxidase complex (Vector Labs) as described previously [8]. Sections are mounted, dehydrated, and coverslipped.
Statistical Analysis
All values are expressed as mean ± standard error of the mean. Comparisons between groups were made with analysis of variance for repeated measures, Student's t test, or GLM analysis of variance where appropriate.
Animal Care
All experimental protocols were preapproved by the Animal Care and Use Committee of the Johns Hopkins Medical Institutions before implementation. Animals received humane care as published in the National Institutes of Health's "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985).
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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). This enhanced activity of NOS in the basal ganglia was blocked in the vicinity of the probe when an NOS inhibitor, L-NAME (1 mmol/L), was infused locally on the contralateral side of the brain (see Fig 1). The difference in NO production with and without inhibitor between contralateral sides of the brain was significant. Using this technique of vehicle in one hemisphere of the brain and vehicle plus drug on the contralateral side, each animal served as its own control.
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). Localization of canine nNOS-containing neurons using lightfield microscopy showed that under normal physiologic conditions, these cells constituted only 1% to 2% of the total neuronal population. After reperfusion following HCA, the lightfield microscopic images demonstrated that the number of nNOS neurons remain few, but heavier staining develops in each neuron (see Fig 2). After HCA and reperfusion, darkfield microscopy of the same images revealed impressive increases in the staining of neuronal processes. These processes amplified and extended from nNOS-containing neurons to pervade all regions of the cortex (see Fig 3). Furthermore, the development of this dense plexus of nNOS fibers was most prominent 20 hours after reperfusion. By 74 hours, the fibers had significantly decreased (see Figs 2, 3). This time-dependent variability in nNOS suggests an induction of nNOS activity after HCA. There was no increase in staining observed when similar canine sections were stained immunocytochemically with monoclonal antibodies against inducible NOS or endothelial NOS (data not shown).
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). This NOS activity was both calcium-dependent and suppressed by analogues of its physiologic substrate, L-arginine. These data support an induced catalytic activity of a calcium-dependent isoform of NOS in the brain that was time-dependent.
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). This increase in NO metabolites reflected a temporal sequence roughly equivalent to the variability of NOS activity demonstrated in Figures 2, 3, and 4
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Recently, NO has been implicated as a mediator of glutamate neurotoxicity via NMDA receptors in a variety of tissues including hippocampal slices [10] and basal ganglial sections [11]. The current mechanism of injury most likely involves an excess of glutamate overstimulating the NMDA receptor, resulting in a large increase in intracellular Ca+2 concentration leading to NOS activation and the production of NO. The NO produced contributes, at least in part, to neuronal death. In addition, NO and glutamate both elicit cell death approximately 12 to 24 hours after insult (a delayed neuronal death), as well as have a concentration-dependent mechanism of action [12].
The most significant finding of our study is that there is a time-dependent variability of NO release during circulatory arrest and reperfusion. This time-dependent NO release was effectively blocked by the NOS inhibitor L-NAME. The induction of NO production was verified using three separate molecular techniques involving both in vivo and in vitro experiments. The first assay involved quantifying NO production in vivo in one cerebral hemisphere while suppressing NO production in the contralateral hemisphere using microdialysis. Because NO has a half-life of only 4 seconds and is produced in low concentrations, we use a microdialysis technique and an in vivo modification of the rapid Bredt-Snyder in vitro assay for NOS activity first developed in 1989 [1, 13]. Although porphyrinic microsensors are available and have been used to detect large changes in NO concentration, their lack of sensitivity made them unsuitable for our study [14]. Microdialysis probes allow local assessment of NO production in specific areas in the brain. In addition, they enable local delivery of drugs to specific regions of the brain without the restriction of the blood-brain barrier. This technique has proved useful because the regional effects of [14C]-L-arginine and L-NAME can be assessed without the confounding variables of a systemic delivery system. Our second technique was an in vitro assay of canine brain tissue homogenates. This is the assay used most frequently by investigators to measure NOS activity, and is the standard method for assessing NO production. This radiolabeled assay measures the amount of arginine that is converted into citrulline, the coproduct of NO. Because NO and citrulline are produced in equimolar concentrations, the amount of citrulline measured infers NO produced. The third method employed was an immunocytochemical assay using commercial monoclonal antibodies to the neuronal-specific isoform of NOS. This assay allows demonstration of nNOS-positive neurons and a qualitative assessment of nNOS staining.
In this study, we also found that the NOS enzyme activity measured was Ca+2/calmodulin-dependent, and canine sections were immunoreactive with monoclonal antibodies directed against nNOS. In the brain, NO is synthesized in neurons, vascular endothelium, perivascular neurons, and astrocytes. Our data suggest that nNOS catalytic activity and expression is being measured, and that it is the neuronal isoform of NOS (not endothelial NOS or inducible NOS) that is increased during HCA and reperfusion. Our data also suggest that expression of the nNOS enzyme is not only constitutive, but also can be induced, especially in the first 24 hours after HCA. This increased expression of NOS activity does not remain elevated but seems to dissipate gradually over time, returning to basal levels 3 days after injury. It is speculative whether this is due to a loss of NOS-positive neurons producing NO or to a reduction of NOS activity after an induced response to a stimulus.
A growing number of results are now suggesting that nNOS expression may be dynamically regulated due to denervation, axotomy, or ischemia [15, 16]. A recent report on focal ischemia showed upregulation of nNOS and its messenger RNA in the cortex and striatum 2 to 48 hours after permanent middle cerebral artery occlusion in Wistar rats [17]. Our laboratory has also observed similar increases in nNOS activity in rats with focal ischemia (Ishiwa, unpublished). Because focal ischemia also induces nNOS and we have failed in preliminary experiments to detect increases in nNOS activity in dogs undergoing normothermic CPB without circulatory arrest, we believe that the ischemic insult is inducing the NOS activity. All control animals had sedation and anesthesia induced and maintained using the same protocol as the experimental group. Neuronal NOS induction was not observed in control canine brains.
Using immunohistochemical staining of canine cerebral sections, we demonstrated that nNOS is expressed in discrete neuronal populations in the normal dog brain, with NOS-containing neurons constituting only 1% to 2% of the neuronal population. During the postreperfusion period, immunohistochemical staining of NOS neurons becomes progressively darker, revealing large, dense networks of fiber plexi. These fibers are widespread and stain heavily for nNOS, suggesting that even with its limited half-life and restricted areas of production, NO may be able to contact large numbers of neurons throughout the cortex. The difference in staining of the darkfield images before and after HCA is marked. Increased NOS staining after glutamate excitotoxicity has also been demonstrated in a culture system [18, 19]. Like the in vivo brain, NOS-containing cultured cortical cells produced a wide distribution of the cell processes during excitotoxicity, suggesting that NOS neurons could mediate widespread neurotoxicity.
NMDA activation has been shown to induce NO synthesis [20]. NMDA receptors have played a significant role in the brain, determining neuronal vulnerability to overstimulation by amino acids after HCA. Inhibition of these NMDA receptors led to improved neurologic outcomes after HCA in our canine survival model [2–4]. Our present results suggest that NO may be an important mediator of amino acid excitotoxicity. Although we have preliminary studies in rats and dogs demonstrating abnormal neuropathology, such as neuronal apoptosis, in the presence of excess neuronal NO, we have been unable to observe improved behavioral outcomes in experimental animals due to the absence of suitable neuronal-specific NO inhibitors.
Further understanding of NO involvement in neurotoxicity after HCA may enable us to understand why some patients, such as the elderly, for example, are more susceptible to neurologic damage than others. The involvement of NO toxicity in neurodegenerative diseases has led to suggestions that advanced age may exhaust buffers to toxic neuronal compounds, rendering individuals more susceptible to injury in later life. Ultimately, understanding the mechanism of neurologic injury after HCA may lead to the development of isoform-specific therapeutic agents that may prove to be beneficial in the clinical setting.
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Acknowledgments |
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Address reprint requests to Dr Brock, Division of Cardiothoracic Surgery, Johns Hopkins Hospital, Blalock 618, 600 North Wolfe St, Baltimore, MD 21287.
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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