Acute regional neuronal injury following hypothermic circulatory arrest in a porcine model
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《血管的通路杂志》
a Department of Cardiac Surgery, Panepistimiou Avenue, Ioannina, 45500 Greece
b Department of Anatomy-Histology-Embryology, University of Ioannina, School of Medicine, Ioannina, Greece
c Department of Cardiac Surgery, Onassis Cardiac Center, Athens, Greece
Presented at the 54th International Congress of the Europen Society for Cardiovascular Surgery, Athens, Greece, May 19–22, 2005.
Abstract
Objective: Although deep hypothermic circulatory arrest (HCA) is routinely used to interrupt normal perfusion of the brain and prevent subsequent cerebral ischemic injury during cardiac surgery, it is associated with various forms of neurologic disturbances. Neurologic sequelae after prolonged HCA include motor, memory and cognitive deficits. The present study was designed to assess acute regional neuronal injury after HCA in an animal model. Methods: Six piglets underwent 75 min of HCA at 18 °C. Four piglets served as normal controls. After gradual rewarming and reperfusion, treatment animals were killed and their brains were perfusion-fixed and cryopreserved. Regional patterns of neuronal apoptosis after HCA was characterized by in situ DNA fragmentation using terminal deoxyneucleotidyl-transferase-mediated biotin-dUTP nick end-labeling (TUNEL) histochemistry. Hematoxylin and eosin histology was used to characterize cell damage morphologically. TUNEL-positive cells were scored on a scale of 0 to 5. Grade 0: no TUNEL-positive cells; Grade 1: <10%, Grade 2: 10–25%, Grade 3: 25–50%, Grade 4: 50–75%; and Grade 5: >75%. Results: TUNEL-positive cells indicating DNA-fragmentation were scored in the precentral gyrus (motor neocortex), postcentral gyrus (sensory neocortex), hippocampus, cerebellum, thalamus and ventral medulla of HCA treated animals and were significantly greater than in normal controls (P0.05). Significantly higher concentrations of TUNEL-positive cells were observed in the sensory and motor neocortex and hippocampus, compared to the cerebellum, thalamus and medulla, indicating an increased selective vulnerability of these brain subregions (P0.05). Despite significant DNA fragmentation indicated by high-concentrations TUNEL-positive cells, no morphologic evidence of apoptosis or necrosis was observed in this acute model. Conclusion: The data indicate that sensory and motor neocortex and hippocampal neurons are selectively vulnerable to neurologic injury after HCA as indicated by elevated levels of TUNEL-positive cells in these brain regions. It is noteworthy that evidence of significant neuronal injury is observed in the acute state. The absence of morphological evidence of apoptosis or necrosis with high levels of TUNEL-positive cells, strongly suggests activation of the apoptotic mechanisms at this early stage. These findings are compatible with data showing morphological evidence of apoptosis in these regions after a more prolonged period in a chronic animal model. The mechanisms underlying neuronal injury, and potential neuroprotective strategies remain to be elucidated.
Key Words: Apoptosis; Neurologic injury; HCA; Hypothermia; TUNEL; Porcine model
1. Introduction
Hypothermic circulatory arrest (HCA) is routinely used to interrupt normal perfusion of the brain and prevent subsequent cerebral ischemic injury during various surgical procedures. Despite its widespread use, persisting concerns remain regarding the optimal implementation of HCA, including the maximal duration and depth of cooling. Although there has been general agreement for more than two decades regarding the overall benefit of profound hypothermia, recently there has been escalating concern about the impact of prolonged durations of HCA on subsequent cognitive function in both adults and in children. Circulatory arrest for longer than 60 min is associated with specific intellectual or motor deficits in adults, and seizures or choreoathetoid movements in small children [1,2].
Recent studies have focused on investigating the mechanisms and cascade of events leading to neuronal injury and eventual nerve cell death, in hopes of finding means for its prevention. Experimental studies have demonstrated that prolonged HCA can lead to neuronal cell death, probably as a consequence of a number of different pathways triggered by ischemia [3–5]. In a number of different animal models, it has been demonstrated that cerebral ischemia (either focal or global) causes neuronal injury by an apoptotic cell death pattern, as well as by necrosis. Ischemia is believed to lead to an imbalance between pro- and anti- apoptotic stimuli, resulting in ongoing tissue injury during and after reperfusion.
Most episodes of hypoxia-ischemia that are severe enough to damage nerve cells, cause variable injury to selected regions, rather than a global injury [6,7]. This results in the clinical patterns of disabilility seen after these insults. This phenomenon, referred to as selective vulnerability, occurs in adult and neonatal brain and reflects heightened sensitivity of specific neuron groups to ischemic injury [1,8]. Previous studies have shown that neurons in the hippocampus, cerebellum, striatum, amygdala, lateral thalamic nucleus, and middle layers of the neocortex are selectively vulnerable to ischemia in adults. Loss of neurons in these regions appears to be, for the most part, responsible for the impairment of memory, cognition, emotional state, and motor function seen in adults after cardiac arrest [4,8,9].
Previous studies suggest that brain damage following HCA is not a uniform process, and that it varies over time [3–5,8–14]. This suggests the need to elucidate the extent of cell damage over time. Although the chronic porcine model of HCA is common in surgical research, many biomedical parameters of early neuronal changes have not been established. In the present study, we sought to characterize acute brain injury after deep HCA in a juvenile pig model to determine the selectively vulnerable cell populations, the patterns of early changes in histopathology, in terms of cell type and injury and the contribution of apoptosis to cell death.
2. Material and methods
Ten male juvenile pigs from a commercial farm, 3–4 months of age and weighing 25–30 kg were used for this study. The animals were divided into two groups: Group A (n=6) underwent hypothermic circulatory arrest at 18 °C for 75 min and Group B (n=4) served as normal control group. All animals were treated humanely and in compliance with the Principles of Laboratory Animal Care, as described by the National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council. The protocol used in this study was also approved by the Animal Care and Use Committee of the University of Ioannina.
2.1. Animal preparation
Catheters were inserted in an ear vein and the left femoral artery for monitoring purposes and withdrawal of blood samples. Anesthesia was induced with intramuscular ketamine hydrochloride (15 mg/kg), atropine (0.05 mg/kg), and dormicum (0.1 mg/kg) and was maintained with intravenous fentanyl (50–200 μg/kg), dormicum and 1–2% isoflurane. Paralysis was achieved with a bolus intravenous rocuronium (0.6 mg/kg) and was maintained with 20% of the total dose every 30 min.
After endotracheal intubation, the animals were ventilated mechanically with 100% oxygen. The ventilator rate and the tidal volume were adjusted to maintain the arterial carbon dioxide tension at 40 mmHg. Blood gas analysis was performed using the alpha-stat method, and the pH during cooling was maintained at 7.4, and carbon dioxide tension at 35–45 mmHg, uncorrected for temperature. Arterial oxygen tension was maintained at greater than 100 mmHg. Hematocrit values during CPB were maintained between 13–23%. A temperature probe was placed in the rectum, while brain temperature was determined with bilateral tympanic membrane probes. Urine output was collected through a bladder catheter (Foley 8–10 F). Arterial pressure, end-expired carbon dioxide, electrocardiogram, and blood gases (ABL Radiometer Medical A/S DK-2700, Copenhagen, Denmark) were monitored.
2.2. Cardiopulmonary bypass and hypothermic circulatory arrest
The chest was opened via a right thoracotomy in the fourth intercostal space. After intervenous heparin (300 IU/kg) was administered, cannulas were advanced to the ascending aorta (16 F arterial cannula) and to the right atrium (single 26 F cannula). Non-pulsatile CPB, using alpha-stat pH management, was initiated at a flow rate of 100 ml/kg per min and then adjusted to maintain a minimum arterial pressure of 50 mmHg. To avoid distension of the left ventricle during CPB, a 10 F vent catheter was inserted via the superior pulmonary vein. After CPB was initiated, the lungs were allowed to collapse.
The CPB circuit included a roller pump, cardiotomy reservoir, arterial filter, water bath and a membrane oxygenator (with integral heat exchanger) (Cobe Cardiovascular Inc, CO, USA) that was primed with a bloodless solution consisting of 1000 cc lactated Ringer's, 50 ml mannitol, and 5000 IU heparin. Sodium bicarbonate was added to adjust the pH to 7.4, as necessary.
After initiation, CPB was continued for approximately 60 min to reach a deep brain temperature of 18 °C in order to ensure a thorough cooling of the brain and to avoid an upward drift of the temperature during the period of circulatory arrest. Myocardial protection was afforded by applying iced saline (4 °C) topically during the 75-min interval of hypothermic circulatory arrest. When the tympanic membrane temperature reached 18 °C, bypass was discontinued, the blood was drained into the oxygenator reservoir, and circulatory arrest was maintained for 75 min. Ice bags were positioned around the head to maintain the brain temperature during HCA. At the end of the arrest, bypass was initiated again with gradual rewarming to a rectal temperature of approximately 35 °C to 36 °C. A temperature gradient exceeding 10 °C between the perfusate and the core temperature was avoided. A temperature of 36 °C was generally reached by approximately 80 min of reperfusion. Minimal doses of inotropic support and crystalloid infusion were used when needed to maintain the systemic pressure above 60 mmHg during reperfusion. Measurements of hemodynamics (heart rate, mean arterial pressure), arterial blood gases, hematocrit, glucose, as well as temperatures were recorded at five time points during the experiment: (1) Baseline at 37 °C and prior to CPB; (2) Before the initiation of CPB; (3) During CPB while cooling to a brain temperature of 18 °C just before HCA; (4) During rewarming to 30 °C; and (5) After CPB.
2.3. Cerebral perfusion/fixation
At the end of the experiment (approximately 160 min after the onset of circulatory arrest), the brain was perfused. A chilled saline solution 0.9% (1 l) followed by 4% paraformaldehyde in 0.1 mol/l phosphate-buffered saline solution (1 l, pH 7.4) were infused into the aortic root after clamping the descending aorta to fix the brain in situ. The descending aorta was cross-clamped to avoid significant loss of perfusion to the lower body. The brains were removed in toto, immersed in 4% paraformaldehyde, and stored at 4 °C in phosphate-buffered saline solution. Control animals (n=4) received no intervention and were put to death for histological analysis.
2.4. Histopathological preparation
All brains were bisected in the sagital plane. Tissue blocks from the left hemisphere were cut to encompass brain regions associated with motor and sensory function, as well as the limbic system and which were known for their vulnerability to hypoxia and ischemia. Brain regions evaluated included the precentral gyrus (motor neocortex), the postcentral gyrus (sensory neocortex), hippocampus, cerebellum, thalamus and anterior ventral medulla. Tissue blocks were dehydrated in ethanol and xylene and embedded in paraffin. Serial 8-μm sections were cut from each tissue block and were mounted onto slides. Hematoxylin and eosin was used to characterize cell damage morphologically. Neuronal apoptosis was characterized by in situ DNA fragmentation using terminal deoxyneucleotidyl-transferase-mediated biotin-dUTP nick end-labeling (TUNEL) histochemistry. The TUNEL assay was performed as described elsewhere [4] with the Apop Tag in situ Apoptosis Detection Kit – Peroxidase (Oncor, Gaithersburg, MD). Each assay included positive and negative control slides.
2.5. Histological evaluation
All slides were evaluated by a neuroanatomist in a blind fashion. Cell damage was categorized as either necrotic or apoptotic according to classic morphologic criteria in sections prepared with hematoxylin and eosin, as previously described [16]. Apoptotic cells were defined by the presence of nuclear karyorrhexis (fragmented, rounded, dense chromatin) and minimal cytoplasmic change, whereas necrotic cells were identified by a pyknotic nucleus or no nucleus (ghost neuron) along with a swollen, eosinophilic cytoplasm. Sections were also evaluated for inflammation, hemorrhage, and infarction.
A semiquantitative scoring system was used to describe the extent of necrotic neuronal injury in the various brain regions. Regions were scored on a scale of 0 to 5 based on the number of damaged neurons in the slices that contained the same areas. The scores were defined as follows: Grade 0, no damaged cells; Grade 1, <10% damaged cells; Grade 2, 10–25% damaged cells; Grade 3, 25–50% damaged cells; Grade 4, 50–75% damaged cells; and Grade 5, >75% damaged cells.
TUNEL (+) cells were identified by a red-stained, condensed nucleus with apoptotic bodies, along with a diminutive or absent cytoplasm. To describe the extent of apoptosis in the various brain regions, a similar semiquantitative scoring system as that described above was used. Each slide was scored on a scale of 0–5, as follows: Grade 0, no TUNEL (+) cells; Grade 1, <10% TUNEL (+) cells; Grade 2,10–25% TUNEL (+) cells; Grade 3, 25–50% TUNEL (+) cells; Grade 4, 50–75% TUNEL (+) cells; and Grade 5, >75% TUNEL (+) cells.
Scores from histological evaluation and TUNEL assays were averaged from 4–8 slides from every region in each animal.
2.6. Statistical analysis
Data are presented as mean and standard deviation (S.D.). Differences among groups were analyzed using analysis of variance (ANOVA) followed by Fisher PSLD post hoc analysis. A value of P0.05 was considered significant.
3. Results
All 6 experimental animals survived the surgical protocol and HCA, as described above. Peri-operative factors, before, during and after HCA are shown in Table 1. The duration of the CPB cooling, CPB warming and total CPB was 57.50±15.75, 82.50±9.46 and 140.00±12.50 min, respectively.
None of the treatment animals undergoing HCA in this acute protocol demonstrated histologic evidence of neuronal damage in any of the brain regions assessed using hematoxylin and eosin staining. In addition, none of the normal control animals demonstrated morphological evidence of neuronal damage as assessed by hematoxylin and eosin staining.
All of the animals undergoing HCA for 75 min at 18 °C demonstrated significantly higher levels of TUNEL positive (+) scores compared to normal controls in all tissue regions examined (Table 2). TUNEL (+) staining was particularly elevated in the neocortex of HCA treated animals compared to control animals (P0.001). In both the sensory and motor neocortex (post- and pre-central gyrus, respectively), TUNEL (+) cells were primarily located in the superficial layers of the gray matter. Normal neurons were found interspersed. TUNEL (+) cells were located in various regions of the hippocampus (P0.05 compared to normal controls), including the CA1–3, CA4 areas and the dentate gyrus. TUNEL scores were also significantly higher in HCA treated animals, compared to normal controls in the anteroventral medulla and the thalamus (P0.05). Animals treated within 75 min of HCA at 18 °C also demonstrated significantly higher scores of TUNEL (+) cells in the cerebellum compared to control animals (P0.05). TUNEL (+) cells were located mostly in the deep granular layer of the cerebellar gray matter.
The various regions examined in the present study also examined different vulnerabilities to the HCA-paradigm used in this study. Among the HCA-treated animals, significantly higher concentrations of TUNEL (+) were observed in the sensory cortex, motor cortex and hippocampus compared to the cerebellum, thalamus and medulla (P0.05 by ANOVA followed by Fisher PSLD). The sensory neocortex demonstrated the highest scores of TUNEL (+) cells compared to other regions. Although not statistically significantly greater than the motor neocortex and hippocampus, the postcentral gyrus (primary sensory neocortex) had much higher TUNEL (+) scores compared to the medulla and thalamus (P0.01) (Fig. 1).
P0.05 vs. sensory and motor cortex, and hippocampus; P0.001 vs. sensory cortex.
4. Discussion
The present study assessed acute neuronal injury in various regions of the brain after HCA in a porcine animal model. We report that neurons in the sensory and motor neocortex, as well as those in the hippocampus, are vulnerable to cell injury acutely after 75 min of HCA at 18 °C, as determined by a positive TUNEL reaction for DNA-fragmentation. Although nerve cell populations in the cerebellum, thalamus and ventral medulla were also found vulnerable to cell injury, the percentage of TUNEL positive cells in these areas was significantly less than that observed in the primary motor and sensory gray matter, and in the hippocampus. These findings are compatible with those reported in chronic models of HCA [3,4,8].
Age and temperature appear to influence neuronal injury, by making certain nerve cell populations more vulnerable to injury [3,6–9]. In particular, the hippocampus, cerebellum, striatum, thalamus, amygdala and neocortex have been reported vulnerable in adult normothermic ischemia [6]. In contrast, newborns were more vulnerable to injury in the neocortex and striatum [7]. Hypothermia does not provide equal protection to all regions of the brain [7]. In adult rats, hypoxia-induced injury in the hippocampus and neocortex is temperature and age-dependent [15]. In the present model of hypothermic ischemia in juvenile pigs, the neocortex and hippocampus demonstrated the greatest vulnerability to insult during HCA. The apparent higher level of TUNEL positive cells in the primary sensory cortex (post-central gyrus) is not clear.
Cerebral injury after HCA is an hypoxia-ischemia induced injury, which results from at least two different pathways, necrosis and apoptosis. Delayed cell death via apoptotic pathways is of special interest because of the potential for intervening in this process. A number of experimental studies have shown that HCA initiates a series of events that ultimately leads to neuronal death via what appears to be a typical apoptotic pattern [3]. A hallmark of apoptosis is the fragmentation of the DNA into smaller ordered oligonucleosomes [16,17]. In the present study, we used the TUNEL assay to detect DNA fragmentation via in situ labeling of the 3-OH end groups. It should be noted, however, that DNA fragmentation could be observed in both apoptosis and necrosis. Moreover, the TUNEL assay has been found to not be completely specific or sensitive [16–19].
Although these previous studies clearly support that some of the cell death observed in HCA is via an apoptotic pathway, the experimental conditions used may underestimate the contribution of apoptosis to the cerebral sequelae after HCA [8]. In this regard, some authors have expressed concern regarding the temporal pattern of brain damage and apoptosis after HCA [3–5,8,11–13]. Thus, although recently improved methods of perfusion-fixation and more sophisticated analysis, have clearly shown the HCA initiates a series of events that ultimately leads to cell death via a typical apoptotic pattern [3,8,19], the time course of these events remains unclear. Most of these previous studies use the classic 90-min HCA, 20 °C model, which results in more severe cerebral injury than that usually observed clinically, where HCA is carried out for shorter intervals [3,8,12,13]. The results from earlier studies also demonstrated that serious cell injury exists as early as 6 h after HCA, and that this process continues for at least 72 h [3,8].
The importance of understanding the time course of events is underscored by an earlier study of long-term survivors of the 90-min, 20 °C protocol [3,12,13]. Although treatment with CsA was reported to improve behavioral recovery after 7 days, at the 7 days time point after HCA there was no difference between CsA treated animals and controls for apoptosis measures [12,13]. The authors concluded that they had missed the peak of apoptosis, and that an effective reduction in nerve cell injury would be found most likely with CsA treatment had they examined brain tissues at an earlier time point.
We found no morphological evidence of apoptosis or necrosis, but significantly greater levels of TUNEL positive cells in the brain regions assessed, compared to normal control animals. We hypothesize, that these findings indicate an early point of activation of the apoptotic pathway. This is consistent with the rapid cell death observed in normal cell suicide programs that can kill a cell within 2 to 3 h. At an earlier time point, such as that in this study, we would not anticipate completion of the apoptotic mechanism, resulting in cell death with its classic morphological characteristics, but rather the initiation of the cellular response cascade. Further studies are required to assess at what point along the apoptotic cascade activation is achieved in this more clinically relevant, acute model.
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b Department of Anatomy-Histology-Embryology, University of Ioannina, School of Medicine, Ioannina, Greece
c Department of Cardiac Surgery, Onassis Cardiac Center, Athens, Greece
Presented at the 54th International Congress of the Europen Society for Cardiovascular Surgery, Athens, Greece, May 19–22, 2005.
Abstract
Objective: Although deep hypothermic circulatory arrest (HCA) is routinely used to interrupt normal perfusion of the brain and prevent subsequent cerebral ischemic injury during cardiac surgery, it is associated with various forms of neurologic disturbances. Neurologic sequelae after prolonged HCA include motor, memory and cognitive deficits. The present study was designed to assess acute regional neuronal injury after HCA in an animal model. Methods: Six piglets underwent 75 min of HCA at 18 °C. Four piglets served as normal controls. After gradual rewarming and reperfusion, treatment animals were killed and their brains were perfusion-fixed and cryopreserved. Regional patterns of neuronal apoptosis after HCA was characterized by in situ DNA fragmentation using terminal deoxyneucleotidyl-transferase-mediated biotin-dUTP nick end-labeling (TUNEL) histochemistry. Hematoxylin and eosin histology was used to characterize cell damage morphologically. TUNEL-positive cells were scored on a scale of 0 to 5. Grade 0: no TUNEL-positive cells; Grade 1: <10%, Grade 2: 10–25%, Grade 3: 25–50%, Grade 4: 50–75%; and Grade 5: >75%. Results: TUNEL-positive cells indicating DNA-fragmentation were scored in the precentral gyrus (motor neocortex), postcentral gyrus (sensory neocortex), hippocampus, cerebellum, thalamus and ventral medulla of HCA treated animals and were significantly greater than in normal controls (P0.05). Significantly higher concentrations of TUNEL-positive cells were observed in the sensory and motor neocortex and hippocampus, compared to the cerebellum, thalamus and medulla, indicating an increased selective vulnerability of these brain subregions (P0.05). Despite significant DNA fragmentation indicated by high-concentrations TUNEL-positive cells, no morphologic evidence of apoptosis or necrosis was observed in this acute model. Conclusion: The data indicate that sensory and motor neocortex and hippocampal neurons are selectively vulnerable to neurologic injury after HCA as indicated by elevated levels of TUNEL-positive cells in these brain regions. It is noteworthy that evidence of significant neuronal injury is observed in the acute state. The absence of morphological evidence of apoptosis or necrosis with high levels of TUNEL-positive cells, strongly suggests activation of the apoptotic mechanisms at this early stage. These findings are compatible with data showing morphological evidence of apoptosis in these regions after a more prolonged period in a chronic animal model. The mechanisms underlying neuronal injury, and potential neuroprotective strategies remain to be elucidated.
Key Words: Apoptosis; Neurologic injury; HCA; Hypothermia; TUNEL; Porcine model
1. Introduction
Hypothermic circulatory arrest (HCA) is routinely used to interrupt normal perfusion of the brain and prevent subsequent cerebral ischemic injury during various surgical procedures. Despite its widespread use, persisting concerns remain regarding the optimal implementation of HCA, including the maximal duration and depth of cooling. Although there has been general agreement for more than two decades regarding the overall benefit of profound hypothermia, recently there has been escalating concern about the impact of prolonged durations of HCA on subsequent cognitive function in both adults and in children. Circulatory arrest for longer than 60 min is associated with specific intellectual or motor deficits in adults, and seizures or choreoathetoid movements in small children [1,2].
Recent studies have focused on investigating the mechanisms and cascade of events leading to neuronal injury and eventual nerve cell death, in hopes of finding means for its prevention. Experimental studies have demonstrated that prolonged HCA can lead to neuronal cell death, probably as a consequence of a number of different pathways triggered by ischemia [3–5]. In a number of different animal models, it has been demonstrated that cerebral ischemia (either focal or global) causes neuronal injury by an apoptotic cell death pattern, as well as by necrosis. Ischemia is believed to lead to an imbalance between pro- and anti- apoptotic stimuli, resulting in ongoing tissue injury during and after reperfusion.
Most episodes of hypoxia-ischemia that are severe enough to damage nerve cells, cause variable injury to selected regions, rather than a global injury [6,7]. This results in the clinical patterns of disabilility seen after these insults. This phenomenon, referred to as selective vulnerability, occurs in adult and neonatal brain and reflects heightened sensitivity of specific neuron groups to ischemic injury [1,8]. Previous studies have shown that neurons in the hippocampus, cerebellum, striatum, amygdala, lateral thalamic nucleus, and middle layers of the neocortex are selectively vulnerable to ischemia in adults. Loss of neurons in these regions appears to be, for the most part, responsible for the impairment of memory, cognition, emotional state, and motor function seen in adults after cardiac arrest [4,8,9].
Previous studies suggest that brain damage following HCA is not a uniform process, and that it varies over time [3–5,8–14]. This suggests the need to elucidate the extent of cell damage over time. Although the chronic porcine model of HCA is common in surgical research, many biomedical parameters of early neuronal changes have not been established. In the present study, we sought to characterize acute brain injury after deep HCA in a juvenile pig model to determine the selectively vulnerable cell populations, the patterns of early changes in histopathology, in terms of cell type and injury and the contribution of apoptosis to cell death.
2. Material and methods
Ten male juvenile pigs from a commercial farm, 3–4 months of age and weighing 25–30 kg were used for this study. The animals were divided into two groups: Group A (n=6) underwent hypothermic circulatory arrest at 18 °C for 75 min and Group B (n=4) served as normal control group. All animals were treated humanely and in compliance with the Principles of Laboratory Animal Care, as described by the National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council. The protocol used in this study was also approved by the Animal Care and Use Committee of the University of Ioannina.
2.1. Animal preparation
Catheters were inserted in an ear vein and the left femoral artery for monitoring purposes and withdrawal of blood samples. Anesthesia was induced with intramuscular ketamine hydrochloride (15 mg/kg), atropine (0.05 mg/kg), and dormicum (0.1 mg/kg) and was maintained with intravenous fentanyl (50–200 μg/kg), dormicum and 1–2% isoflurane. Paralysis was achieved with a bolus intravenous rocuronium (0.6 mg/kg) and was maintained with 20% of the total dose every 30 min.
After endotracheal intubation, the animals were ventilated mechanically with 100% oxygen. The ventilator rate and the tidal volume were adjusted to maintain the arterial carbon dioxide tension at 40 mmHg. Blood gas analysis was performed using the alpha-stat method, and the pH during cooling was maintained at 7.4, and carbon dioxide tension at 35–45 mmHg, uncorrected for temperature. Arterial oxygen tension was maintained at greater than 100 mmHg. Hematocrit values during CPB were maintained between 13–23%. A temperature probe was placed in the rectum, while brain temperature was determined with bilateral tympanic membrane probes. Urine output was collected through a bladder catheter (Foley 8–10 F). Arterial pressure, end-expired carbon dioxide, electrocardiogram, and blood gases (ABL Radiometer Medical A/S DK-2700, Copenhagen, Denmark) were monitored.
2.2. Cardiopulmonary bypass and hypothermic circulatory arrest
The chest was opened via a right thoracotomy in the fourth intercostal space. After intervenous heparin (300 IU/kg) was administered, cannulas were advanced to the ascending aorta (16 F arterial cannula) and to the right atrium (single 26 F cannula). Non-pulsatile CPB, using alpha-stat pH management, was initiated at a flow rate of 100 ml/kg per min and then adjusted to maintain a minimum arterial pressure of 50 mmHg. To avoid distension of the left ventricle during CPB, a 10 F vent catheter was inserted via the superior pulmonary vein. After CPB was initiated, the lungs were allowed to collapse.
The CPB circuit included a roller pump, cardiotomy reservoir, arterial filter, water bath and a membrane oxygenator (with integral heat exchanger) (Cobe Cardiovascular Inc, CO, USA) that was primed with a bloodless solution consisting of 1000 cc lactated Ringer's, 50 ml mannitol, and 5000 IU heparin. Sodium bicarbonate was added to adjust the pH to 7.4, as necessary.
After initiation, CPB was continued for approximately 60 min to reach a deep brain temperature of 18 °C in order to ensure a thorough cooling of the brain and to avoid an upward drift of the temperature during the period of circulatory arrest. Myocardial protection was afforded by applying iced saline (4 °C) topically during the 75-min interval of hypothermic circulatory arrest. When the tympanic membrane temperature reached 18 °C, bypass was discontinued, the blood was drained into the oxygenator reservoir, and circulatory arrest was maintained for 75 min. Ice bags were positioned around the head to maintain the brain temperature during HCA. At the end of the arrest, bypass was initiated again with gradual rewarming to a rectal temperature of approximately 35 °C to 36 °C. A temperature gradient exceeding 10 °C between the perfusate and the core temperature was avoided. A temperature of 36 °C was generally reached by approximately 80 min of reperfusion. Minimal doses of inotropic support and crystalloid infusion were used when needed to maintain the systemic pressure above 60 mmHg during reperfusion. Measurements of hemodynamics (heart rate, mean arterial pressure), arterial blood gases, hematocrit, glucose, as well as temperatures were recorded at five time points during the experiment: (1) Baseline at 37 °C and prior to CPB; (2) Before the initiation of CPB; (3) During CPB while cooling to a brain temperature of 18 °C just before HCA; (4) During rewarming to 30 °C; and (5) After CPB.
2.3. Cerebral perfusion/fixation
At the end of the experiment (approximately 160 min after the onset of circulatory arrest), the brain was perfused. A chilled saline solution 0.9% (1 l) followed by 4% paraformaldehyde in 0.1 mol/l phosphate-buffered saline solution (1 l, pH 7.4) were infused into the aortic root after clamping the descending aorta to fix the brain in situ. The descending aorta was cross-clamped to avoid significant loss of perfusion to the lower body. The brains were removed in toto, immersed in 4% paraformaldehyde, and stored at 4 °C in phosphate-buffered saline solution. Control animals (n=4) received no intervention and were put to death for histological analysis.
2.4. Histopathological preparation
All brains were bisected in the sagital plane. Tissue blocks from the left hemisphere were cut to encompass brain regions associated with motor and sensory function, as well as the limbic system and which were known for their vulnerability to hypoxia and ischemia. Brain regions evaluated included the precentral gyrus (motor neocortex), the postcentral gyrus (sensory neocortex), hippocampus, cerebellum, thalamus and anterior ventral medulla. Tissue blocks were dehydrated in ethanol and xylene and embedded in paraffin. Serial 8-μm sections were cut from each tissue block and were mounted onto slides. Hematoxylin and eosin was used to characterize cell damage morphologically. Neuronal apoptosis was characterized by in situ DNA fragmentation using terminal deoxyneucleotidyl-transferase-mediated biotin-dUTP nick end-labeling (TUNEL) histochemistry. The TUNEL assay was performed as described elsewhere [4] with the Apop Tag in situ Apoptosis Detection Kit – Peroxidase (Oncor, Gaithersburg, MD). Each assay included positive and negative control slides.
2.5. Histological evaluation
All slides were evaluated by a neuroanatomist in a blind fashion. Cell damage was categorized as either necrotic or apoptotic according to classic morphologic criteria in sections prepared with hematoxylin and eosin, as previously described [16]. Apoptotic cells were defined by the presence of nuclear karyorrhexis (fragmented, rounded, dense chromatin) and minimal cytoplasmic change, whereas necrotic cells were identified by a pyknotic nucleus or no nucleus (ghost neuron) along with a swollen, eosinophilic cytoplasm. Sections were also evaluated for inflammation, hemorrhage, and infarction.
A semiquantitative scoring system was used to describe the extent of necrotic neuronal injury in the various brain regions. Regions were scored on a scale of 0 to 5 based on the number of damaged neurons in the slices that contained the same areas. The scores were defined as follows: Grade 0, no damaged cells; Grade 1, <10% damaged cells; Grade 2, 10–25% damaged cells; Grade 3, 25–50% damaged cells; Grade 4, 50–75% damaged cells; and Grade 5, >75% damaged cells.
TUNEL (+) cells were identified by a red-stained, condensed nucleus with apoptotic bodies, along with a diminutive or absent cytoplasm. To describe the extent of apoptosis in the various brain regions, a similar semiquantitative scoring system as that described above was used. Each slide was scored on a scale of 0–5, as follows: Grade 0, no TUNEL (+) cells; Grade 1, <10% TUNEL (+) cells; Grade 2,10–25% TUNEL (+) cells; Grade 3, 25–50% TUNEL (+) cells; Grade 4, 50–75% TUNEL (+) cells; and Grade 5, >75% TUNEL (+) cells.
Scores from histological evaluation and TUNEL assays were averaged from 4–8 slides from every region in each animal.
2.6. Statistical analysis
Data are presented as mean and standard deviation (S.D.). Differences among groups were analyzed using analysis of variance (ANOVA) followed by Fisher PSLD post hoc analysis. A value of P0.05 was considered significant.
3. Results
All 6 experimental animals survived the surgical protocol and HCA, as described above. Peri-operative factors, before, during and after HCA are shown in Table 1. The duration of the CPB cooling, CPB warming and total CPB was 57.50±15.75, 82.50±9.46 and 140.00±12.50 min, respectively.
None of the treatment animals undergoing HCA in this acute protocol demonstrated histologic evidence of neuronal damage in any of the brain regions assessed using hematoxylin and eosin staining. In addition, none of the normal control animals demonstrated morphological evidence of neuronal damage as assessed by hematoxylin and eosin staining.
All of the animals undergoing HCA for 75 min at 18 °C demonstrated significantly higher levels of TUNEL positive (+) scores compared to normal controls in all tissue regions examined (Table 2). TUNEL (+) staining was particularly elevated in the neocortex of HCA treated animals compared to control animals (P0.001). In both the sensory and motor neocortex (post- and pre-central gyrus, respectively), TUNEL (+) cells were primarily located in the superficial layers of the gray matter. Normal neurons were found interspersed. TUNEL (+) cells were located in various regions of the hippocampus (P0.05 compared to normal controls), including the CA1–3, CA4 areas and the dentate gyrus. TUNEL scores were also significantly higher in HCA treated animals, compared to normal controls in the anteroventral medulla and the thalamus (P0.05). Animals treated within 75 min of HCA at 18 °C also demonstrated significantly higher scores of TUNEL (+) cells in the cerebellum compared to control animals (P0.05). TUNEL (+) cells were located mostly in the deep granular layer of the cerebellar gray matter.
The various regions examined in the present study also examined different vulnerabilities to the HCA-paradigm used in this study. Among the HCA-treated animals, significantly higher concentrations of TUNEL (+) were observed in the sensory cortex, motor cortex and hippocampus compared to the cerebellum, thalamus and medulla (P0.05 by ANOVA followed by Fisher PSLD). The sensory neocortex demonstrated the highest scores of TUNEL (+) cells compared to other regions. Although not statistically significantly greater than the motor neocortex and hippocampus, the postcentral gyrus (primary sensory neocortex) had much higher TUNEL (+) scores compared to the medulla and thalamus (P0.01) (Fig. 1).
P0.05 vs. sensory and motor cortex, and hippocampus; P0.001 vs. sensory cortex.
4. Discussion
The present study assessed acute neuronal injury in various regions of the brain after HCA in a porcine animal model. We report that neurons in the sensory and motor neocortex, as well as those in the hippocampus, are vulnerable to cell injury acutely after 75 min of HCA at 18 °C, as determined by a positive TUNEL reaction for DNA-fragmentation. Although nerve cell populations in the cerebellum, thalamus and ventral medulla were also found vulnerable to cell injury, the percentage of TUNEL positive cells in these areas was significantly less than that observed in the primary motor and sensory gray matter, and in the hippocampus. These findings are compatible with those reported in chronic models of HCA [3,4,8].
Age and temperature appear to influence neuronal injury, by making certain nerve cell populations more vulnerable to injury [3,6–9]. In particular, the hippocampus, cerebellum, striatum, thalamus, amygdala and neocortex have been reported vulnerable in adult normothermic ischemia [6]. In contrast, newborns were more vulnerable to injury in the neocortex and striatum [7]. Hypothermia does not provide equal protection to all regions of the brain [7]. In adult rats, hypoxia-induced injury in the hippocampus and neocortex is temperature and age-dependent [15]. In the present model of hypothermic ischemia in juvenile pigs, the neocortex and hippocampus demonstrated the greatest vulnerability to insult during HCA. The apparent higher level of TUNEL positive cells in the primary sensory cortex (post-central gyrus) is not clear.
Cerebral injury after HCA is an hypoxia-ischemia induced injury, which results from at least two different pathways, necrosis and apoptosis. Delayed cell death via apoptotic pathways is of special interest because of the potential for intervening in this process. A number of experimental studies have shown that HCA initiates a series of events that ultimately leads to neuronal death via what appears to be a typical apoptotic pattern [3]. A hallmark of apoptosis is the fragmentation of the DNA into smaller ordered oligonucleosomes [16,17]. In the present study, we used the TUNEL assay to detect DNA fragmentation via in situ labeling of the 3-OH end groups. It should be noted, however, that DNA fragmentation could be observed in both apoptosis and necrosis. Moreover, the TUNEL assay has been found to not be completely specific or sensitive [16–19].
Although these previous studies clearly support that some of the cell death observed in HCA is via an apoptotic pathway, the experimental conditions used may underestimate the contribution of apoptosis to the cerebral sequelae after HCA [8]. In this regard, some authors have expressed concern regarding the temporal pattern of brain damage and apoptosis after HCA [3–5,8,11–13]. Thus, although recently improved methods of perfusion-fixation and more sophisticated analysis, have clearly shown the HCA initiates a series of events that ultimately leads to cell death via a typical apoptotic pattern [3,8,19], the time course of these events remains unclear. Most of these previous studies use the classic 90-min HCA, 20 °C model, which results in more severe cerebral injury than that usually observed clinically, where HCA is carried out for shorter intervals [3,8,12,13]. The results from earlier studies also demonstrated that serious cell injury exists as early as 6 h after HCA, and that this process continues for at least 72 h [3,8].
The importance of understanding the time course of events is underscored by an earlier study of long-term survivors of the 90-min, 20 °C protocol [3,12,13]. Although treatment with CsA was reported to improve behavioral recovery after 7 days, at the 7 days time point after HCA there was no difference between CsA treated animals and controls for apoptosis measures [12,13]. The authors concluded that they had missed the peak of apoptosis, and that an effective reduction in nerve cell injury would be found most likely with CsA treatment had they examined brain tissues at an earlier time point.
We found no morphological evidence of apoptosis or necrosis, but significantly greater levels of TUNEL positive cells in the brain regions assessed, compared to normal control animals. We hypothesize, that these findings indicate an early point of activation of the apoptotic pathway. This is consistent with the rapid cell death observed in normal cell suicide programs that can kill a cell within 2 to 3 h. At an earlier time point, such as that in this study, we would not anticipate completion of the apoptotic mechanism, resulting in cell death with its classic morphological characteristics, but rather the initiation of the cellular response cascade. Further studies are required to assess at what point along the apoptotic cascade activation is achieved in this more clinically relevant, acute model.
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