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Eur J Cardiothorac Surg 2005;27:74-80
© 2005 Elsevier Science NL

Cooling to 10°C and treatment with Cyclosporine A improve cerebral recovery following prolonged hypothermic circulatory arrest in a chronic porcine model

Justus T. Straucha,*, David Spielvogela, Peter L. Haldenwanga, Ning Zhanga, Donald Weisza, Carol A. Bodianb, Nadine A. Tattonc, Randall B. Grieppa

a Departments of Cardiothoracic Surgery, Neurosurgery, Mount Sinai School of Medicine, New York University, New York, NY, USA
b Biomathematics, Mount Sinai School of Medicine, New York University, New York, NY, USA
c Neurology, Mount Sinai School of Medicine, New York University, New York, NY, USA

Received 8 August 2004; received in revised form 21 October 2004; accepted 25 October 2004.

* Corresponding author. Address: Department of Cardiothoracic and Vascular Surgery, Friedrich-Schiller-University of Jena, Erlanger Allee 101, 07747 Jena, Germany. Tel.: +49 3641 9322901; fax: +49 3641 9322902. (E-mail: ju.strauch{at}gmx.de).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Objective: This study was undertaken to assess whether cooling to 10°C and/or treatment with Cyclosporine A (CsA) can reduce neurological injury during prolonged hypothermic circulatory arrest (HCA) in a chronic animal model. Methods: In this blinded study, 24 pigs (20–23kg) were randomized to HCA for 90min at 20°C (n=8), at 10°C (n=8), or at 10°C with 5mg/kg CsA (n=8). CsA (or placebo) were given intravenously before and for 3 days after HCA. Hemodynamics and neurophysiological data were monitored periodically throughout the experiment and for 3h after HCA, as well as intracranial pressure (ICP), which has been shown to correlate with outcome. Daily neurological/behavioral evaluation (mental status, coordination and appetite; 12=normal and 0=coma or death) was carried out until sacrifice on postoperative day (POD) 3. Results: Overall survival rate was 83.3%: one 20°C control, two 10°C controls, and one 10°C/CsA pig died and were replaced. Basic hemodynamic data revealed no significant differences between groups. ICP differed significantly among the groups during the first 3h postoperatively (P=0.003 by repeated measures ANOVA); it was higher in the 20°C group than in the 10°C/CsA or 10°C control groups. Recovery of visual evoked potentials was significantly better in the10°C/CsA group than in the 10°C control group; no recovery was seen by 3h in the 20°C control group. Postoperative behavioral scores also differed significantly between the groups, P=0.03: a good behavioral outcome—a score >9 on POD3—was more prevalent among CsA-treated pigs (75%) than among 10°C controls (50%), or 20°C controls (12.5%, P=0.06). Conclusions: The data suggest that cooling to 10°C and CsA treatment are both of benefit in improving cerebral recovery after HCA when compared with untreated 20°C controls, and may be synergistic.

Key Words: Cerebral protection • Extracorporeal circulation • Great vessels


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Hypothermic circulatory arrest (HCA) is one of the methods routinely used to prevent cerebral ischemic injury during operations requiring interruption of normal perfusion to the brain: during repair of lesions involving the aortic arch, or for correction of various congenital heart lesions in infancy. There is, however, persisting concern about the safety and optimal implementation of HCA: the maximal safe duration, optimal depth of cooling, and management of reperfusion. It has been shown that periods of prolonged HCA are associated with long-lasting cognitive impairment in both adults and in children, and in experimental studies, prolonged HCA can lead to neuronal cell death, probably as a consequence of a number of different pathways triggered by ischemia [1–3].

To allow experimental validation of approaches to improving cerebral protection involving HCA, an animal model is required in which cerebral physiology as well as behavioral responses and histopathological findings can be studied [4]. From such studies, it has been documented that HCA is accompanied by increased capillary permeability and by inflammatory responses triggered by cardiopulmonary bypass that may synergistically aggravate ischemic brain injury [5]. Animal studies have also led to the observation that small but consistent increases in ICP accompany neuronal injury following HCA, and may contribute to it [6]. Other experimental studies have reported that apoptosis, in addition to necrosis, may contribute to brain injury after ischemia [3,7] and that apoptotic pathways may be reversible in their earlier stages [8,9].

The recognition that neuronal cell death following ischemia may involve apoptosis [9–11] introduces new and exciting possibilities in terms of pharmacological strategies to increase cerebral tolerance to HCA. The opportunity to prevent initiation of apoptotic cell death following HCA by preoperative—and therefore pre-ischemic— treatment, augmented by ongoing therapy during reperfusion, makes this strategy more enticing in this context than its appeal for other situations involving ischemia, such as stroke. In a study using a chronic porcine model, we found that cyclosporine A (CsA), an immunosuppressive agent widely used to reduce rejection after transplantation and to treat autoimmune disorders, had a favorable impact on HCA-induced injury in the brain [3,9]. Although we were attracted to this drug because of reports that it had the potential to reduce apoptosis, we were unable to demonstrate that this was the mechanism of its action, possibly because sacrifice of the animals was carried out long after the probable peak of apoptosis. We speculated that CsA may also act by influencing the inflammatory response of the body to ischemia and reperfusion, and/or by blocking other cell-death pathways.

Experimental studies have also indicated that cooling to more profound hypothermia than the 20°C used in our standard protocol can also enhance cerebral protection, and reduce neuronal cell death following HCA. We therefore hypothesized that the combination of CsA and profound hypothermia might act synergistically to reduce ischemic cerebral injury. Thus, the present study was undertaken to assess whether cooling to 10°C and/or treatment with cyclosporine A (CsA) at 10°C can reduce neurological injury during prolonged periods of HCA in a chronic animal model, compared with less profoundly cooled, untreated controls. Hemodynamic, neurophysiological and neurobehavioral assessments of recovery after HCA were carried out. After 3 days, the brains were harvested for detailed evaluation of the possible effect of CsA and/or low temperature on apoptosis.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1. Study design
A blinded study was initiated to compare the effects of CsA and low temperature during a period of HCA on cerebral metabolism, behavior and apoptosis in the hippocampus at 3 days after HCA. Twenty-four female juvenile Yorkshire pigs (Th. D. Morris, Inc., Reisterstown, NY, USA), 2–3 months of age, weighing 20–23kg, underwent 90min of hypothermic circulatory arrest (HCA) at two different brain temperatures. The animals were randomized to HCA for 90min at 20°C without drug treatment (n=8); HCA for 90min at 10°C without drug treatment (n=8), and HCA for 90min at 10°C plus treatment with 5mg/kg CsA (Novartis Pharmaceuticals Co., East Hanover, NJ, USA) (n=8), given intravenously before and after HCA, as well as subcutaneously for 3 days postoperatively. Randomization was carried out prior to the start of the protocol by an independent member of the Department of Biomathematics, and was revised after half of the animals had been studied to allow compensation for animals that died prior to completion of all the required examinations.

Each animal underwent preoperative neurological behavior examination, intraoperative hemodynamic and metabolic monitoring, and recording of quantitative electroencephalogram (EEG), quantitative visual evoked potentials (VEP) and cervical as well as cortical somatosensory evoked potentials (SSEP) until 3h after HCA. Postoperative detailed daily gross behavioral/neurological assessment was performed until elective sacrifice on postoperative day (POD) 3. For histopathological assessment, all brains were selectively perfused and analyzed by two different methods for a quantitative evaluation of the amount of apoptosis in the hippocampus.

2.2. Perioperative management and anesthesia
All animals received humane care in compliance with the guidelines ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals’ published by the National Institute of Health (NIH Publication No. 88-23, revised 1985). The protocols for all experiments were approved by the Mount Sinai Institutional Animal Care and Use Committee.

After pretreatment with intramuscular ketamine (15mg/kg), and atropine (0.03mg/kg), animals were anesthetized with intravenous sodium thiopenthal (20mg/kg). Following endotracheal intubation, the pigs were ventilated mechanically with a FiO2 of 0.5 and isoflurane 1–2% to maintain deep anesthesia. Paralysis was achieved with intravenous pancuronium (0.1mg/kg). The ventilator rate and the tidal volume were adjusted to maintain the arterial carbon dioxide tension at about 35–40mmHg. End-expiratory carbon dioxide, and inspiratory and expiratory isoflurane were monitored continuously (PPG Biomedical Systems, Model 2010-200 R, Lenexa, KS, USA). Before intervention and after all incisions were closed, all animals received cephazoline (15mg/kg) intravenously.

A bladder catheter (Foley 10F) was inserted for online measurement of urine output and temperature probes were placed in the esophagus, rectum and the brain via a small burr hole in the skull. An arterial line was placed in the left femoral artery for pressure monitoring and blood sampling (pH, oxygen tension, carbon dioxide tension, oxygen saturation, base excess, hematocrit, hemoglobin, glucose and lactate; Blood Gas Analyzer, Ciba Corning 865, Chiron Diagnostics, Norwood, MA, USA). A thermodilution catheter (Baxter Healthcare Corp., Irvine, CA, USA) for assessment of cardiac output was inserted in the femoral vein and advanced into the pulmonary artery.

2.3. Electrophysiological evaluation
2.3.1. Electroencephalograpy (EEG)
As described previously [2], the periosteum was removed to expose the sagittal and coronal sutures of the calvarium via a midline incision. Five stainless steel screw electrodes were drilled in the skull 2.0centimeters to the left and right of the sagittal suture. The reference and ground electrode was placed over the frontal sinus. Electrodes were connected to a Spectrum 32 EEG machine (Spectrum 32, Cadwell Laboratories Inc., Kennewick, WA, USA), where the signal was amplified, filtered, digitized, and stored on an optical disk for subsequent analysis. At each measurement timepoint, 5min of continuous EEG was recorded. Because the prognostic value of the EEG is hampered by the use of anesthetic drugs, isoflurane was adjusted to 1% in all animals.

2.3.2. Somatosensory evoked potential recording (SSEP)
Somatosensory evoked potentials (SSEPs) were recorded from the cervical spine and contralateral parietal cortex in response to stimulation of the left and right median nerves. Amplifiers were set at a 5000 gain with bandpass filters settings of 30–1000Hz for cervical responses and 10–300Hz for cortical responses. Electrical stimulation (25mA, 0.1ms duration) of the median nerve was delivered through a pair of stainless steel needle electrodes that were inserted at the most distal joint on the posterior surface of each forelimb.

The cervical recording electrode was a long stainless steel needle that was insulated except at the tip (impedance <3000{Omega}). The electrode was inserted at approximately the second cervical vertebra and lowered until the spine was contacted. The most repeatable potential from the cervical site was a negative–positive wave. The latency of the negative wave was 6–8ms, and the latency of the positive peak was 8–10ms.

Using a nose reference, a large negative–positive complex could be recorded from electrodes placed at skull sites overlying the parietal cortex. The peak latency of the negative potential was 17–19ms. Two sets of SSEPs were recorded for each median nerve at each of the measurement timepoints. Each waveform was obtained by averaging the responses to 500 stimuli. The latencies and amplitudes were measured for the positive cervical potential at 7–9ms and the negative cortical potential at 19–21ms for all animals.

2.3.3. Visual evoked potential recording (VEP)
The method of recording VEPs has been described in detail elsewhere [12]. Briefly, VEPs were elicited from the pig by a photo stimulator (model SLS 4100), providing a flash intensity of 1J and a stimulation frequency of 1Hz. Only the left eye was stimulated in each animal for the VEP recordings. The experiments were performed in a darkened room with the flashlight at a constant distance of 40cm from the left eye.

The analog data were recorded on a rectilinear pen recorder and were also digitized and stored in a computer system for later analysis. Initially, we confirmed that the VEP implicit times and amplitudes were stable, and then recorded and stored them as baseline values (measurement 1). No recording was made prior to initiation of HCA, but subsequent recordings were carried out during rewarming and recovery, in conjunction with hemodynamic measurements as indicated below.

2.3.4. Intracranial pressure (ICP)
For continuous intracranial pressure monitoring, a parenchymal pressure probe was inserted via a small burr hole in the skull and connected to a transducer (Codman ICP ExpressTM, Johnson and Johnson Prof. Inc., Raynham, MA, USA).

2.3.5. Operative technique
The chest was opened via a small right thoracotomy in the fourth intercostal space. After heparinization (300IU/kg) the ascending aorta was cannulated with a 16F arterial cannula, and the right atrium with a single 26F cannula. Non-pulsatile CPB, using alpha–stat pH management, was initiated at a flow rate of 100ml/kg/min and then adjusted to maintain a minimum mean arterial pressure of 50mmHg. To avoid distension of the left ventricle during CPB, a 10F vent catheter was inserted via the superior pulmonary vein. After initiation of CPB, the lungs were allowed to collapse. A heat exchanger was utilized for core cooling, and surface cooling was achieved using a cooling blanket.

The cardiopulmonary bypass circuit included roller pumps, a cardiotomy reservoir, and a membrane oxygenator (VPCML Plus, Cobe Cardiovascular Inc., Arvada, CO, USA) which was primed with a bloodless solution consisting of 1000cc 0.9% NaCl, furosemide (1mg/kg), heparin (5000IU) and KCl (1.5meq/kg). After initiation, CPB was continued for 45min to reach a deep brain temperature of 20°C or 10°C, and to insure thorough cooling to avoid an upward drift of the temperature during the period of circulatory arrest. The operating room temperature during HCA was maintained at 18—20°C. In all animals, myocardial protection during the 90-min interval of HCA was afforded by topical cooling, using iced saline (~4°C) in the pericardium.

Thereafter, CPB was reinstituted, and core and surface rewarming were begun and continued to an esophageal temperature of approximately 35–36°C. Care was taken to avoid a temperature difference between the perfusate and core temperature of more than 10°C. During weaning from CPB, 3–5mg/kg/min dobutamine was frequently utilized. In cases with low vascular resistance, norepinephrine was occasionally necessary.

All pigs received intramuscular cephalosporine (15mg/ kg) as well as pain medication (buterophenol 0.1mg/kg) for the first 3 postoperative days, and were kept in separated pens for the entire observation period, with a 12-h light-dark cycle, and a room temperature of 22–24°C. Animals that refused liquids orally during the first 24h were treated intravenously with 500ml of Ringer's solution over 8h. If solids were refused from the bowl, hand-feeding was carried out.

2.3.6. Study protocol
Measurements of hemodynamics (heart rate, central venous pressure, mean arterial pressure, pulmonary arterial pressure and cardiac output), arterial blood gases, hematocrit, glucose and lactate, as well as temperatures, EEG, VEP and SSEP were recorded at 6 timepoints during the experiments.

1. at baseline at 37°C, prior to CPB
2. during CPB at 20 or 10°C—lowest brain temperature—prior to HCA
3. during rewarming, at 30°C
4. one hour after the start of rewarming
5. two hours after the start of rewarming
6. three hours after the start of rewarming

Prior to CPB, pigs received in a blinded fashion either CsA 2.5mg/kg (n=8), or placebo (50% castor oil, 50% olive oil) (n=16), both diluted in 100ml 0.9% NaCl, over a 10-min interval. Hemodynamic measurements were performed at 5-min intervals for 15min to study possible drug side-effects. Another 2.5mg/kg dose of CsA or placebo was given 1h after CPB in the same fashion. Postoperatively, all animals received daily doses of 5mg/kg CsA or placebo subcutaneously until elective sacrifice on POD 3.

2.3.7. Behavioral score
From POD 1 to POD 3, all animals were scored on a gross behavioral grading scale that evaluated mental status, appetite, and coordination, by a physician blinded to the experimental protocol. Daily scores were determined after several time-standardized inspections in the morning, at noon and in the evening. A score of 12 points is normal, and 0 indicates coma or death (see Table 1).


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  		Table 1. Behavioral score
 
2.3.8. Histological assessment
Brains were perfusion-fixed in situ using buffered formaldehyde, with the head packed in ice. All brains were bisected in the sagittal plane and a tissue block encompassing the left hippocampus was infiltrated with sucrose and processed for subsequent histological analysis as previously described [8]. Apoptotic nuclei were identified by bright green fluorescence of the nucleic acid-binding cyanine dye (YOYO) in a typical chromatin condensation pattern, combined with bright red in situ end labeling (ISEL) fluorescence, indicating DNA fragmentation [9,13].

The histological evaluation of the hippocampus was carried out by a neurobiologist with extensive experience evaluating apoptosis who was blinded to the experimental intervention. The definition of apoptotic cell death and so-called ‘ischemic’ and ‘necrotic’ cell death pathways has been extensively described elsewhere [9].

2.3.9. Statistical methods
All animals were randomized to control and treatment groups by an independent party, who also coded and labeled all doses of CsA and placebo. Groups were compared separately at baseline, during CPB, and after CPB. The t-test or the Mann–Whitney test, as appropriate, was used for comparisons at baseline. When the data were consistent with normality and equal variance assumptions, the measurements during CPB as well as those after CPB were compared using repeated measures ANOVA, with tests for average differences between groups and for group–time interactions (change in the difference between groups over time).

Otherwise the groups were compared separately at each time point using the Mann–Whitney or Fisher exact tests. We report p values unadjusted for multiple testing: their purpose is not for an exact global assessment but rather as a guide to help interpret the pattern of differences between groups at different times. The Bonferroni correction was not utilized because we expect these tests at successive time points to be highly correlated. Analyses were implemented with SAS software on a VAX computer and StatXact 4 for Windows.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1. Mortality and morbidity
The overall survival rate in all three groups was 83.3%. One 10°C/CsA-treated animal (12.5%), one placebo-treated 20°C animal (12.5%), and two 10°C/placebo animals (25%) died before elective sacrifice on POD 3, and were replaced in a blinded fashion. Of the animals that did not survive, the 10°C/CsA-treated animal died intraoperatively of intractable ventricular arrhythmia, and the remaining pigs succumbed to lung problems.

3.2. Comparability of experimental groups
All study animals were female, 3–4 months old, and housed for a period of more than 2 days in the Department of Comparative Medicine and Surgery of the Mount Sinai Medical Center. A comparison of the preoperative animal weights (10°C/CsA: 21±2.0kg versus 10°C/placebo: 22.5±3.2kg versus 20°C/placebo: 20.5±2.5kg) and age showed no differences between the groups.

Basic hemodynamic data showed some minor variations, but no clinically relevant differences between groups in heart rate, mean arterial pressure, central venous pressure, pulmonary artery pressure, or cardiac output. Acid–base, blood gas and metabolic parameters are shown in Table 2, and also revealed no unexpected differences among the groups. Lactate levels were significantly higher following HCA at 20°C than at 10°C.


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  		Table 2. Acid–base, blood gas and metabolic parameters
 
3.3. Intracranial pressure (ICP)
There were no significant differences in baseline ICP values between the groups. In all three groups, there was a clear decrease in ICP during cooling. During reperfusion after weaning from CPB, there was an increase in intracranial pressure in all the groups, but ICP was significantly lower (P=0.01) over the whole 3h observation period after HCA in both 10°C groups compared with the group which underwent HCA at 20°C. CsA treatment failed to lower the ICP further than in the placebo group (see Fig. 1). A detailed comparison of ICP between 10°C placebo and 20°C placebo pigs revealed no significant difference after 1h of recovery, but a significantly lower ICP after 2h (P=0.01) and 3h (P=0.001) in the 10°C placebo group.



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  		Fig. 1. Average differences in direct measurements of intracranial pressure (ICP) after 90min of hypothermic circulatory arrest (HCA) at 10°C in pigs treated with cyclosporine A (CsA, n=8), placebo (n=8), or at 20°C with placebo (n=8).

 
3.4. Electrophysiological recovery
EEG recovery, ranging from 4 to 25% of baseline values 3h after HCA, was observed in 6/8 CsA-treated animals, in 8/8 of the 10°C placebo group, but in only 4/8 of the 20°C pigs. The highest percentage of EEG recovery was reached—a mean of 10.6% of baseline—by the 10°C placebo animals; 10°C/CsA group pigs showed recovery to 4.4% of baseline EEG activity 3h after discontinuing bypass, and the 20°C animals reached only 3.9% of baseline. Although somewhat higher in animals with HCA at 10°C, the rate of EEG recovery failed to differ significantly among the three study groups (P=0.06).

Cortical somatosensory evoked potentials showed somewhat better recovery in the CsA group, but this did not reach statistical significance.

Results for testing visual evoked potential recovery 3h after discontinuing CPB showed the highest values for the 10°C/CsA group: recovery was 42.3% of baseline. This was significantly higher (P=0.03) than the 25.2% recovery of VEPs of the 10°C placebo-treated animals. VEPs in the 20°C placebo-treated animals did not recover by 3h after discontinuing CPB (see Fig. 2).



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  		Fig. 2. Recovery of visual evoked potentials (VEP) for the first 3h after the start of rewarming, following 90min of hypothermic circulatory arrest (HCA). Values are mean % recovery, with standard deviation.

 
3.5. Metabolic parameters
All three groups showed a slightly higher lactate after cooling to 10°C or 20°C on CPB than at baseline. There was a marked increase in arterial lactate levels during the first hour of reperfusion after the 90min of HCA and completion of rewarming in all three groups: the highest levels occurred in the 20°C placebo animals, with values significantly higher than in the two 10°C groups. Over the ensuing hours of recovery, all groups showed a progressive decline in their lactate levels, but none of the groups reached baseline values after 3h of recovery.

3.6. Behavioral score
Postoperative behavioral scores differed significantly between the groups, as seen in Fig. 3, P=0.03. A good behavioral outcome—a score >9 on POD3—was more prevalent among CsA-treated pigs (75%) than among 10°C controls (50%), or 20°C controls (12.5%, P=0.06).



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  		Fig. 3. Group comparison for behavioral scores after recovery from 90min of hypothermic circulatory arrest (HCA). POD, postoperative day.

 
3.7. Histopathological evaluation
The median number of apoptotic nuclei in serial sections of the hippocampus failed to show any significant reduction in 10°C CsA-treated or 10°C placebo-treated animals compared with animals cooled only to 20°C: the mean was 1.05±0.8 apoptotic cells/mm3 in 10°C CsA-treated animals, 1.89±2.2 apoptotic cells/mm3 in 10°C animals without drug treatment and 1.14±0.7 apoptotic cells/mm3 in controls cooled down to 20°C.

The count for other forms of cell nuclear changes—T1-cells—representing ischemic chromatin changes, also showed no significant differences: the mean was 0.5±0.7 T1cells/mm3 in 10°C CsA-treated animals, 0.93±0.8T1cells/mm3 in 10°C animals without drug treatment, and 0.54±0.7T1cells/mm3 in controls cooled to 20°C.

The count for nuclei representing necrotic cells—T2-cells—revealed somewhat greater differences among the groups, but these differences were not statistically significant. The lowest level was seen in 10°C CsA-treated animals: the mean was 0.007±0.02T2cells/mm3 in 10°C CsA-treated animals, 0.73±0.8T2cells/mm3 in 10°C animals without drug treatment, and 0.32±0.5T2cells/mm3 in controls cooled down to 20°C.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
The simplest strategy for cerebral protection during aortic surgery and repair of some types of congenital heart disease is still the use of hypothermic circulatory arrest (HCA). Although there has been general agreement for more than two decades regarding the benefit and relative safety of profound hypothermia, there is escalating concern about the impact of the prolonged durations of HCA sometimes required for these procedures on subsequent cognitive function in both adults and in children [14]. This has prompted a search for improved ways of implementing HCA as well as alternatives to its use. At present, however, there is no consensus about the optimal temperature conditions for maximizing cerebral protection during HCA, or the value of additional treatment with neuroprotective substances [15].

This study has confirmed earlier findings in our laboratory suggesting that treatment with the neuroprotective agent cyclosporine A (CsA) and use of very profound hypothermia each result in a neurological outcome superior to what is seen with more conventional implementation of HCA. Moreover, this study suggests that the effects of these two strategies may be synergistic. The chronic porcine model of HCA used in this study, which allows evaluation of multiple indices of cerebral recovery, provides reliable data that is highly relevant to clinical practice.

The mechanisms responsible for the effect of CsA in reducing ischemia-induced injury are not fully understood. Although CsA is primarily known for its efficacy as an immunosuppressive agent, it was chosen for evaluation in this context because of its potential for reducing apoptosis. In the current study, which was designed to allow evaluation of postoperative behavioral recovery for three days, we may again have missed the optimal time for detection of apoptosis, and may therefore have obscured a beneficial effect of CsA via this mechanism, as discussed in a previous report [3]. On the other hand, the failure to find a significant reduction in apoptosis in treated animals at 72h in this study—as well as in pilot studies using CsA at 20°C—may mean that inhibition of apoptosis is not an important mechanism by which CsA exerts its neuroprotective influence. It is interesting that CsA did have a more marked impact in lowering the incidence of necrotic cell death in this as in the earlier study (in which CsA was used during HCA at 20°C.) But differences in the overall levels of apoptosis found in this study compared with previous investigations by the same neuropathologist—possibly due to differences in the efficacy of fixation despite use of the same protocols—make us feel that these histopathological results may not be definitive, and that further studies of the role of apoptosis and other forms of cell death following HCA—with and without CsA—are still needed.

Although this study does not help in elucidating the mechanism, it does affirm that CsA exerts a neuroprotective effect if given before and following HCA. It has recently been demonstrated [16] that immunological reactions play an important role in ischemia-reperfusion injury in the brain, and CsA may act by inhibiting this process. In the present study, we were able to show significantly lower ICP in the CsA-treated animals as well as in the more profoundly cooled animals throughout reperfusion and early recovery following HCA [17]. The lower ICP in CSA-treated and colder animals correlated with evidence of more rapid VEP recovery and with consistently higher behavioral scores for the first 3 days after HCA compared with placebo-treated animals cooled to 20°C. This study thus adds to the evidence that lower ICP is associated with more rapid and complete cerebral recovery following hypothermic cerebral protection strategies, but it does not clarify whether cerebral edema is actually involved in the pathogenesis of cerebral injury after HCA, or whether higher ICP is merely a diagnostic sign of more severe ischemic damage.

Since a number of studies have demonstrated that cerebral metabolism is reduced more effectively at more profound levels of hypothermia, it seems logical that cerebral protection should be greater when HCA is carried out colder [18,19]. There has been some reluctance to implement this idea clinically because it involves more prolonged cooling and rewarming and because of fear that truly profound cooling may cause problems in other organ systems, including coagulopathy and capillary leak syndrome [13,20,21]. The current study adds to the evidence that more profound cooling—to 10°C—does not have a major adverse systemic impact, and that it does result in better behavioral recovery.

The data clearly indicate that treatment with CsA and cooling to profound hypothermic conditions—10°C—result in better cerebral protection than either strategy alone. The evidence is quite consistent—including electrophysiological parameters of recovery, histopathology, and behavioral scores—but is particularly striking with regard to recovery of VEPs [22]. VEPs show more rapid and superior recovery than other evoked potential responses like SSEPs, and this study suggests that use of VEPs may prove to be a reliable, non-invasive and easy-to-implement online monitoring tool for future experimental studies of cerebral protection.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
This study adds to the evidence that cerebral protection may be better during HCA at very cold temperatures than with more moderate hypothermia. The results also confirm an earlier study suggesting that pre- and post-ischemic treatment with CsA improve neurological recovery after deep hypothermic circulatory arrest in a porcine model, and suggest that the effect of CsA and deep hypothermia may be synergistic. The neuroprotective impact of CsA has now been demonstrated in two separate studies at different temperatures, and should perhaps prompt consideration of clinical trials of the impact of cyclosporine A on cerebral recovery in high-risk patients undergoing prolonged HCA.


   Acknowledgments
 
This study was supported by grant HL 45636 from the National Institutes of Health.

We would like to thank Richard Henry for technical assistance and Russell Jenkins for his care of the animals.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

  1. Midulla PS, Gandsas A, Sadeghi AM, Mezrow CK, Yerlioglu ME, Wang W, Wolfe D, Ergin MA, Griepp RB. Comparison of retrograde cerebral perfusion to antegrade cerebral perfusion and hypothermic circulatory arrest in a chronic porcine model. J Card Surg 1994;9:560-575.[Medline]
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