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Ann Thorac Surg 1997;64:1082-1088
© 1997 The Society of Thoracic Surgeons
Departments of Cardiothoracic Anesthesia and Pediatric and Congenital Heart Surgery, The Cleveland Clinic Foundation, Cleveland, Ohio, and the Departments of Anesthesiology, Cardiothoracic Surgery, and Pathology, Tufts University Schools of Medicine and Veterinary Medicine, Boston, Massachusetts
Accepted for publication April 17, 1997.
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Abstract |
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Methods. Neonatal lambs (weight, 4 to 6 kg) anesthetized with isoflurane were supported by cardiopulmonary bypass, subjected to 90 or 120 minutes of HCA at 15°C, and rewarmed on bypass to 38°C. One hour after cardiopulmonary bypass was terminated, the brains were perfusion fixed and removed for in situ hybridization and immunohistochemical analysis. Some animals survived 3 days before their brains were removed to examine for neuronal necrosis. One group of lambs (n = 20) received aptiganel (2.5 mg/kg). A second group (n = 25) received saline vehicle only.
Results. Increasing duration of HCA induced a corresponding increase in c-fos messenger RNA expression throughout the hippocampal formation and cortex. However, Fos protein synthesis peaked after 90 minutes of HCA and decreased significantly (p < 0.01) after 120 minutes of HCA. Aptiganel administration caused a significant decrease in (p < 0.001) c-fos messenger RNA expression and Fos protein synthesis after 90 minutes of HCA and preserved Fos protein synthesis after 120 minutes of HCA. Neuronal necrosis was observed in the brains of vehicle-treated lambs after 120 minutes of HCA but was significantly decreased (p < 0.05) in the lambs given aptiganel.
Conclusions. These experiments indicate that the transcriptional processes of immediate-early genes remain intact, whereas translational processes are impaired after prolonged HCA. The inability to synthesize Fos proteins after 120 minutes of HCA was associated with neuronal degeneration. Aptiganel preserved translational processes and caused a significant improvement in the neurologic outcome.
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Introduction |
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The underlying mechanisms responsible for causing the neurologic complications accompanying cardiopulmonary bypass (CPB) and hypothermic circulatory arrest (HCA) are poorly understood. However, recent animal experiments have provided evidence that the mechanism of injury from CPB and HCA involves the same excitotoxic processes that are thought to be at work in stroke, traumatic brain injury, subdural hemorrhage, and global ischemia [1, 2].
The cellular damage that occurs after ischemic or traumatic brain injury is believed to result from the excessive release of the excitatory neurotransmitter L-glutamate and its actions at the N-methyl-D-aspartate (NMDA) and non-NMDA receptors [3]. The NMDA receptor is both a voltage- and ligand-gated ion channel whose normal function is to mediate synaptic transmis-sion. Glutamate binds to a specific site on the NMDA receptor, allowing cations to cross the cell membrane. In pathologic conditions, such as ischemia, trauma, hypoxia, or seizures, the excessive intracellular accumulation of cations, particularly calcium, is believed to be a key step in the development of NMDA receptor–mediated neuronal injury [4]. The intracellular calcium accumulation in turn initiates the expression of rapidly induced transcriptional activators known as the immediate-early genes. The best-studied intermediate-early gene, c-fos, is expressed within minutes in the hippocampal formation of the brain after seizures, hypoxia, and global ischemia in response to increased intracellular calcium concentrations [4]. The protein product of c-fos messenger RNA (mRNA), Fos protein, is an intracellular signaling molecule capable of modulating the transcription of several late-response genes, including p53, heat-shock protein, bcl-x, tyrosine hydroxylase, and opiate peptide genes [5–7]. Whereas some of the late-response genes expressed after c-fos induction are associated with apoptosis, others enhance cell survival [5, 7]. Therefore the appearance of nuclear-associated Fos protein has become a useful indicator of severely stressed neurons and provides an effective method for assaying pharmacologic interventions. We have previously demonstrated a relationship between an increasing duration of HCA and expression of the immediate-early gene c-fos, with subsequent neuronal degeneration in the hippocampus, a region of the brain important in memory and cognitive functions and especially vulnerable to injury from CPB and HCA [1].
Aptiganel hydrochloride (CNS 1102; Cerestat) is a noncompetitive NMDA antagonist that has been shown to be neuroprotective in in vitro and in vivo models of excitotoxic neuronal injury, including traumatic brain injury and cerebral ischemia [8, 9]. Aptiganel binds to a site within the ion channel pore of the NMDA receptor, thereby blocking the ion channel and preventing calcium ions from entering the cell. In the following experiments we determined the effects of aptiganel, administered before CPB and HCA, on the expression of c-fos mRNA, the intranuclear expression of Fos-related protein, and delayed neuronal degeneration in the hippocampal formation of lambs. The purpose of these experiments was to test the hypothesis that aptiganel would provide neuroprotection during HCA.
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Experimental Design and Methods |
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Forty-five neonatal lambs (average age, 10 days; average weight, 5.9 kg) were anesthetized by mask with isoflurane. After endotracheal intubation, the lungs were mechanically ventilated with 100% oxygen and 2% isoflurane. Animals were paralyzed with vecuronium (0.1 mg/kg intravenously). The electrocardiogram, end-tidal CO2, inspired and expired isoflurane concentration, and oxygen saturation were monitored in all animals. Temperature was monitored using temporalis muscle, esophageal, and rectal thermistors and maintained at 38°C before and after CPB with a heating blanket. A femoral arterial catheter was placed for continuous blood pressure monitoring and blood sampling.
Cardiopulmonary bypass was achieved by direct cannulation of the right atrium with a 16F or 18F venous cannula (Bard, USCI, Tewksbury, MA) placed through a right thoracotomy incision and the descending aorta with an 8F or 10F arterial cannula (Bard, USCI) through the right femoral artery. The CPB circuit included a Minimax pediatric membrane oxygenator (Medtronics, Anaheim, CA), a Medtronic pediatric arterial line filter, a CDI inline arterial/venous blood gas monitor (3M Healthcare, Ann Arbor, MI), and a Stockert (Munich, Germany) roller pump. The pump prime consisted of 500 mL of fresh whole sheep blood, 100 mg of SoluCortef (hydrocortisone sodium succinate), 25 mEq sodium bicarbonate, mannitol (0.5 g/kg), furosemide (0.25 mg/kg), 1,500 units of heparin, and 300 mg of CaCl2.
Heparin (300 U/kg) was administered intravenously before the animals were cannulated for CPB. The activated clotting time was monitored with a Hemochron 400 (International Technidyne Corp, Edam, NJ). One group of animals (n = 20) received aptiganel (1.25 mg/kg, intravenously; Cambridge Neuroscience, Boston, MA) 5 to 10 minutes before the right atrium was cannulated. This dose was determined from previously performed pharmacokinetic studies in lambs. A second group of animals (n = 25) received saline vehicle. Arterial blood gas, electrolyte, and glucose concentrations were determined before, during, and after CPB and corrected as indicated. No intravenous solutions containing glucose were given at any time throughout the experiment. Balanced salt solutions were administered at a rate of 4 mL • kg-1 • h-1. Blood losses were replaced with fresh whole sheep blood.
After CPB was initiated, animals were cooled to 14° to 16°C by surface and core cooling. All animals had their heads packed in ice. The CPB pump was turned off at 16° to 18°C, and HCA was maintained for 90 minutes in 22 animals (14 vehicle treated and 8 aptiganel treated) and 120 minutes in 23 animals (11 vehicle treated and 12 aptiganel treated). Animals were rewarmed on CPB to 38°C. Approximately 10 minutes before CPB was terminated, the animals in the aptiganel-treated group received a second dose of aptiganel (1.25 mg/kg intravenously). Once normothermia was achieved, the lambs were weaned from CPB.
A control group (n = 6) received general anesthesia with 1% to 2% isoflurane for 3 hours but did not have CPB. Three more animals were anesthetized with isoflurane and then given 100 mg/kg of pentylenetetrazol as an intravenous bolus to induce seizure. The pentylenetetrazol seizure model is used to induce the expression of c-fos and Fos-related antigens in the hippocampus [7].
One hour after CPB was terminated, 24 animals (13 vehicle treated and 11 aptiganel treated) were killed with a bolus of KCl. The brains were perfusion fixed with 1,000 mL of chilled, heparinized saline, followed by 1,000 mL of chilled 4% paraformaldehyde, and removed. The remaining animals (11 vehicle treated and 10 aptiganel treated) were weaned from mechanical ventilation, extubated, and allowed to recover for 3 days. Surviving animals received intercostal nerve blocks with 0.5% bupivacaine before the chest was closed. The time to achieve major milestones after operations such as extubation, standing, bleating, and nursing, was recorded. An ovine coma scale, described later, was used to compare groups. After 3 days the animals in the survival groups were anesthetized again by mask with isoflurane before they were killed with KCl and their brains fixed. Brains were removed and embedded in paraffin, after which 5-µm-thick coronal sections were cut and stained with hematoxylin-eosin. With the aid of a light microscope, a veterinary neuropathologist blinded to the treatment groups counted the number of dead neurons in the hippocampal formation. The dead neurons were counted in the same regions of the hippocampal formation as were used for the in situ and immunohistochemical analyses.
In Situ Hybridization and Immunohistochemistry
Brains from the acute experiments were cut into 12-µm-thick coronal sections and thaw-mounted onto gelatin-coated slides. Plasmids containing either sense or antisense reading frames were transcribed using SP6 polymerase according to previously described procedures [10]. Sections were hybridized at 55°C overnight, treated with RNAase to eliminate nonspecifically bound probe, and stringently washed at 55°C in 0.1x standard saline citrates. Slides were apposed to x-ray film for 1 week, then dipped in NTB2 and exposed for 2 to 3 weeks. Only the cells and tissue exclusively labeled with the antisense probe represented specific hybridization for c-fos mRNA. Sections were stained with thionine to locate the specific regions of the hippocampal formation and then examined under dark-field illumination at x20 magnification to quantify the Fos-encoding mRNA.
Immunohistochemical analyses were performed as previously described [1, 11]. Briefly, brains cut into 12-µm-thick coronal sections were pretreated with 0.1% H2O2 in methanol for 20 minutes to eliminate endogenous peroxidase activity (ie, blood cells). Slide-mounted sections were incubated with primary antibody for c-fos and Fos-related proteins (generously donated by Dr. Iadarola; final dilution 1:2,000). This peptide sequence is common to three members of the FOS family: fra-1, fra-b, and c-fos. The sections were incubated with biotinylated goat antirabbit serum and processed by the avidin-biotin-peroxidase method (Vector, Burlingame, CA), using diaminobenzidine as the peroxidase substrate. This procedure was modified to include nickel intensification of the diaminobenzidine reaction. Mounted and coverslipped tissue sections were examined under a Zeiss microscope at x20 magnification. The finding of the intranuclear brown-black reaction product indicated the presence of immobilized antigen.
Using a previously described image-processing system, a blinded observer detected and quantified c-fos mRNA and intranuclear Fos-like immunoreactivity [11]. Neurons in the hippocampal formation were easily distinguished from glia, ependyma, and choroid cells by their characteristic pyramidal morphology, as well as their size and distribution.
Neurologic Injury Evaluation
All animals in the survivor groups were assessed neurologically using a modification of the ovine behavioral scale developed by Dr Swain's laboratory [12]. The neurologic deficit scoring consisted of five major components, including level of consciousness, motor and sensory function, feeding behavior, and vocalization. A score of 10 was given for normal function and 0 for brain death. Final neurologic deficits were agreed upon by at least two members of the team.
Data Analysis
All values are expressed as the mean ± the standard error of the mean. In situ hybridization and immunohistochemical data were evaluated for the treatment groups using the Kruskal-Wallis one-way analysis of variance on the ranks. Study groups were compared using Dunn's method (InStat program). A p value of less than 0.05 was considered statistically significant.
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Results |
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TopFootnotesAbstractIntroductionExperimental Design and MethodsResultsCommentAcknowledgmentsReferences |
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The control animals (n = 6; no CPB), which were given only isoflurane general anesthesia, were found to have very low concentrations of c-fos mRNA in the hippocampus and cortex. There was no Fos-like immunoreactivity protein expression in any neurons in the hippocampus or cortex of control animals. The pentylenetetrazol-treated lambs (n = 3) had very high concentrations of c-fos mRNA and intense intranuclear Fos-like immunoreactivity in neurons throughout the hippocampus and cortex, similar to that seen in rodents [7].
Cardiopulmonary bypass with increasing duration of HCA induced a corresponding increase in the expression of c-fos mRNA in the hippocampal formation, with maximal expression after 120 minutes of HCA (n = 6) (Fig 1
) in vehicle-treated lambs. Fos-like immunoreactivity was greatest after 90 minutes of HCA and decreased significantly after 120 minutes of HCA in vehicle-treated lambs (Figs 2, 3). The intensity of intranuclear Fos staining after 90 minutes of HCA (n = 7) was similar to that observed in the pentylenetrazol-treated animals.
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) to be significantly inhibited after 90 minutes of HCA in all regions of the hippocampal formation and cerebral cortex examined. The amount of the in situ hybridization signal and protein staining (n = 6) in the drug-treated animals after 90 minutes of HCA was similar to that observed in control animals receiving general anesthesia alone without CPB or HCA. After 120 minutes of HCA, c-fos mRNA expression was observed in aptiganel-treated animals in all regions of the hippocampus, but was still less than that observed in the vehicle-treated animals (see Fig 1). Fos-like immunoreactivity protein expression was significantly greater in the aptiganel-treated animals (n = 5) than in the vehicle-treated group (n = 6) after 120 minutes of HCA (p < 0.01) (see Fig 3). In other words, the immediate-early gene response observed in the vehicle-treated animals after 90 minutes of HCA was not observed in aptiganel-treated animals until after 120 minutes of HCA.
Aptiganel-treated animals were significantly slower (p < 0.05) to wake up and took longer to wean from mechanical ventilation than vehicle-treated animals, averaging 8.5 hours to extubation compared with 5.5 hours in vehicle-treated animals. However, aptiganel-treated animals had significantly better pulmonary function and functional recovery in the early postoperative periods, particularly those in the 90-minute HCA group. Four of the 5 aptiganel-treated animals subjected to 120 minutes of HCA had uneventful recoveries and, unlike the vehicle-treated lambs, were ambulating and nursing on postoperative day 1 (p < 0.05) (Fig 4). Only 1 of the vehicle-treated lambs in the 120-minute HCA group could ambulate or nurse from its mother on postoperative day 3. All of the aptiganel-treated animals were observed to have nystagmus up to 12 hours after operation.
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). Necrosis was not seen in the dentate gyrus neurons in either group.
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Comment |
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TopFootnotesAbstractIntroductionExperimental Design and MethodsResultsCommentAcknowledgmentsReferences |
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A wide range of therapeutic strategies have been investigated, with the intention of improving outcome, but few have shown convincing clinical benefit. Recent studies have shown that the mechanisms of injury from CPB and HCA may involve the same excitotoxic processes at work in stroke, prolonged seizures, traumatic head injury, subdural hemorrhage, perinatal hypoxia, and global ischemia [15]. All of these conditions involve the release of glutamate and other excitatory neurotransmitters, leading to excessive excitation of the NMDA and non-NMDA receptors and the unregulated entry of calcium and sodium into the neuron (see [15] for review). The rise in the intracellular free calcium concentration initiates a cascade of events involving gene expression, enzyme induction, and free radical release, which eventually produces neuronal injury and death [3–7, 15].
Recently introduced neuroprotective therapy for cerebral ischemia attempts to interrupt this cascade of events that leads to neuronal death. Aptiganel is a noncompetitive NMDA receptor antagonist that protects neurons by binding to a site within the calcium channel. It has been shown to reduce cerebral infarct volume in animals [8, 9]. In preliminary reports of stroke patients given aptiganel, those who received the highest dose showed a statistically significant improvement in neurologic outcome at 90 days as compared with the outcome in the placebo group [16].
Administering aptiganel before an ischemic event such as that produced by HCA is a logical application. Our experiments have shown that aptiganel pretreatment leads to a reduction in the neuronal degeneration in the hippocampus that occurs after 2 hours of HCA in neonatal lambs. Aptiganel also appears to delay the immediate-early gene response by approximately 30 minutes. That is, the intense intranuclear Fos protein expression (without neuronal necrosis) observed in vehicle-treated animals after 90 minutes of HCA did not occur in the aptiganel-treated animals until after 120 minutes of HCA (see Fig 3). Furthermore, these experiments demonstrated that the inability to synthesize Fos-related proteins in the brains of the vehicle-treated animals after 2 hours of HCA, presumably as a result of hypoxia, acidosis, and depleted energy stores, is associated with neuronal degeneration. Other animal stroke models have shown an association between the postischemic inhibition of Fos protein synthesis in CA1 pyramidal cells and their subsequent death [1, 6, 17]. Greeley and associates [18] reported their finding of a delayed recovery of cerebral metabolism and mitochondrial oxidation after HCA. Functioning mitochondria are essential for protein translation to occur. Further studies are needed to determine which late-response genes and gene products activated by Fos protein lead to apoptosis or cell survival.
Aptiganel-treated animals had significantly better pulmonary function (ie, better arterial blood gas values) and less pulmonary edema after 90 minutes of HCA than the vehicle-treated groups did. It is not clear from these experiments whether the pulmonary edema resulted from an inflammatory response, cardiac failure, or a centrally mediated (neurogenic) mechanism after HCA. However, there was no hemodynamic indication of cardiac failure. Furthermore, NMDA receptors are presumed to be present only in the central nervous system, and this, together with the absence of pulmonary edema in the aptiganel-treated animals, supports a neurogenic mechanism. Using an isolated perfused and ventilated rat lung model, Said and colleagues [19] induced pulmonary edema with glutamate and prevented it with the noncompetitive NMDA antagonist MK-801. We have found an association between the development of pulmonary edema and an intense Fos-related protein response in the brain after 90 minutes of HCA, which was prevented with aptiganel. The absence of this response after 120 minutes of HCA correlates with the brain's inability to translate Fos-related proteins, and therefore other late-response genes and gene products that may mediate the lung injury. We are not aware of other reports of improved pulmonary function after CPB and HCA with NMDA antagonists. Presumably this effect would apply to other organs as well.
This study has demonstrated the efficacy of aptiganel in reducing the neurologic injury associated with prolonged HCA. Aptiganel-treated animals showed significantly better pulmonary function and functional recovery after HCA and significantly less necrosis of CA1 and CA3 hippocampal neurons. Expression of the immediate-early genes associated with cellular stress and injury was delayed by aptiganel, thereby potentially extending the safe time limit for HCA.
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Acknowledgments |
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TopFootnotesAbstractIntroductionExperimental Design and MethodsResultsCommentAcknowledgmentsReferences |
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Footnotes |
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TopFootnotesAbstractIntroductionExperimental Design and MethodsResultsCommentAcknowledgmentsReferences |
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References |
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TopFootnotesAbstractIntroductionExperimental Design and MethodsResultsCommentAcknowledgmentsReferences |
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