**7. Experimental investigation of cerebral injury following DHCA**

A clearer understanding of the consequences of HCA will be pivotal in clinical decisionmaking, including when to initiate circulatory arrest and the appropriate interval. Delayed cell death is of special interest because of the potential for intervention. Although apoptosis is believed to play a part in the cerebral injury, its role has generally been identified through histologic techniques. These snapshots do not permit a clear delineation of the time-line of apoptosis. Because its role is not clear, therapies have yet to be designed for the specific purpose of inhibiting apoptosis.

The balance between cell survival and death is under tight genetic control (Almeida et al., 2000). A multiplicity of extracellular signals and intracellular mediators are involved in maintaining this balance. When the cell is exposed to physical, biochemical or biological injury, or deprived of necessary substances, it activates a series of stress-response genes. Although with minimal insults, the cell may recover, with greater insults, single cell death results. The current understanding of the neurons response to insult has been supported by evidence from a series of studies using a porcine model system to investigate the effects hypothermic circulatory arrest and ischemic insult on the integrity of neuronal populations.

### **7.1 Clinically relevant animal models**

Evaluating various strategies and treatments in animal studies in order to determine clinical feasibility remains a challenge. Animal models have contributed immensely to our understanding of cerebral consequences of HCA, with several animal models having been used. To date, the preclinical investigation of cerebral injury mechanisms related to deep hypothermic circulatory arrest has been limited to large-animal models (porcine, canine and ovine). These models are expensive, personnel demanding, cumbersome and are usually performed without validated neuropsychologic assessment. Rodent models have been attempted to overcome some of these disadvantages, although treatment effects cannot always be confirmed in the rat model. Ultimately, however, each experimental model system from cell cultures to rats, to large animals and ultimately to clinical trials, have their advantages and disadvantages, and ultimately their place in these investigations.

Most animal models require an extended period of arrest to produce a reproducible level of neuronal injury that would facilitate elucidating the mechanisms of injury and efficacy of neuroprotective interventions. Many large animal models require DHCA for at least 90-120 minutes to demonstrate neurologic deficits. Although such prolonged DHCA interval might not be considered clinically realistic, they may be more appropriate for demonstrating the molecular pathways behind acute neuronal injury and hence, modes of intervention (Conti et al, 1998).

Study of a neuroprotective strategy includes appropriate selection of an animal model and functional indices. The model is selected with respect to their relevance and feasibility of assessing the parameters of interest. Investigations of promising neuroprotectvie methods require validation (validation study), use in experimental settings to optimize cerebral protection during CPB and DHCA (performance study) and test during routine cardiac surgery (clinical study).

Hypothermia is essential for cerebral protection during HCA. Hypothermia reduces cerebral metabolic activity, oxygen demand, and prevents the release of neurotransmitters and delays the onset of fatal biochemical cascade (Elrich et al., 2002; McCullough et al., 1999). Although reduced, brain metabolism is not suppressed adequately and remains

A clearer understanding of the consequences of HCA will be pivotal in clinical decisionmaking, including when to initiate circulatory arrest and the appropriate interval. Delayed cell death is of special interest because of the potential for intervention. Although apoptosis is believed to play a part in the cerebral injury, its role has generally been identified through histologic techniques. These snapshots do not permit a clear delineation of the time-line of apoptosis. Because its role is not clear, therapies have yet to be designed for the specific

The balance between cell survival and death is under tight genetic control (Almeida et al., 2000). A multiplicity of extracellular signals and intracellular mediators are involved in maintaining this balance. When the cell is exposed to physical, biochemical or biological injury, or deprived of necessary substances, it activates a series of stress-response genes. Although with minimal insults, the cell may recover, with greater insults, single cell death results. The current understanding of the neurons response to insult has been supported by evidence from a series of studies using a porcine model system to investigate the effects hypothermic circulatory arrest and ischemic insult on the integrity of neuronal populations.

Evaluating various strategies and treatments in animal studies in order to determine clinical feasibility remains a challenge. Animal models have contributed immensely to our understanding of cerebral consequences of HCA, with several animal models having been used. To date, the preclinical investigation of cerebral injury mechanisms related to deep hypothermic circulatory arrest has been limited to large-animal models (porcine, canine and ovine). These models are expensive, personnel demanding, cumbersome and are usually performed without validated neuropsychologic assessment. Rodent models have been attempted to overcome some of these disadvantages, although treatment effects cannot always be confirmed in the rat model. Ultimately, however, each experimental model system from cell cultures to rats, to large animals and ultimately to clinical trials, have their

Most animal models require an extended period of arrest to produce a reproducible level of neuronal injury that would facilitate elucidating the mechanisms of injury and efficacy of neuroprotective interventions. Many large animal models require DHCA for at least 90-120 minutes to demonstrate neurologic deficits. Although such prolonged DHCA interval might not be considered clinically realistic, they may be more appropriate for demonstrating the molecular pathways behind acute neuronal injury and hence, modes of intervention (Conti

Study of a neuroprotective strategy includes appropriate selection of an animal model and functional indices. The model is selected with respect to their relevance and feasibility of assessing the parameters of interest. Investigations of promising neuroprotectvie methods require validation (validation study), use in experimental settings to optimize cerebral protection during CPB and DHCA (performance study) and test during routine cardiac

Hypothermia is essential for cerebral protection during HCA. Hypothermia reduces cerebral metabolic activity, oxygen demand, and prevents the release of neurotransmitters and delays the onset of fatal biochemical cascade (Elrich et al., 2002; McCullough et al., 1999). Although reduced, brain metabolism is not suppressed adequately and remains

advantages and disadvantages, and ultimately their place in these investigations.

**7. Experimental investigation of cerebral injury following DHCA** 

purpose of inhibiting apoptosis.

**7.1 Clinically relevant animal models** 

et al, 1998).

surgery (clinical study).

relatively high at 18°C in traditional HCA protocols (McCullough et al., 1999). In light of evidence suggesting that the apoptotic pathway may be reversible in their earlier stages (McCullough et al., 1999), studies from our team were undertaken to assess whether cooling to 10°C can reduce neurological injury during 75 minutes of HCA in an acute porcine model compared to less profoundly cooled (18°C) animals, as assessed by DNA fragmentation, anti-apoptotic protein Bcl-2 expression, and ultrastructural changes in the sensory cortex. Sixteen male juvenile pigs from a commercial farm, 2-3 months of age and weighing 25-35 Kg were used for this study. The animals were divided into three groups: Group A (*n*=6) underwent hypothermic circulatory arrest at 18oC for 75 min, Group B (*n*=6) underwent hypothermic circulatory arrest at 10oC for 75 min and Group C (*n*=4) served as normal controls.

Preparation and surgery were performed as previously described (Ananiadou et al 2005). Briefly, catheters were inserted in an ear vein and the left femoral artery for monitoring purposes and withdrawal of blood samples. Anesthesia was induced with intramuscularly 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% to 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.

Animals were ventilated mechanically with 100% oxygen, after endotracheal intubation. Ventilator rate and tidal volume were adjusted to maintain the arterial carbon dioxide tension at 40 mmHg. Hematocrit values during cardiopulmonary bypass (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.

As previously described, the chest was opened via a right thoracotomy in the fourth intercostal space (Ananiadou et al., 2005). After administration of intravenous heparin (300 IU/kg), cannulas were advanced to the ascending aorta (16 F arterial cannula) and to the right atrium (single 26 F cannula). Non-pulsatile CPB, 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. The lungs were allowed to collapse after CPB was initiated. The CPB circuit was primed with a bloodless solution consisting of 1000cc lactated Ringer's, 50 ml mannitol, and 5000 IU heparin. Sodium bicarbonate was added to adjust the pH to 7.4, as necessary.

CPB was continued for an average 58 or 106 minutes, to reach a deep brain temperature of 18oC or 10oC, respectively. Myocardial protection was afforded by applying iced saline (4oC) topically during the 75-minute interval of hypothermic circulatory arrest. When the tympanic membrane temperature reached 18oC or 10oC, bypass was discontinued, the blood was drained into the oxygenator reservoir, and circulatory arrest was maintained for 75 minutes. 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 35oC to 36oC. A temperature gradient exceeding 10oC between the perfusate and the core temperature was avoided. A temperature of 36oC was reached after an average of 83 or 104 minutes of reperfusion for animals treated with 18 oC

Neurologic Injury Following Hypothermic Circulatory Arrest 251

Neuronal death is normal during development of the nervous system, but it is abnormal in brain and spinal cord disease and injury. The available evidence indicates that the survival of neurons and their death are highly regulated and finely orchestrated dynamic events that depend on a number of internal and external factors. Two types of cell death are recognized: cell necrosis resulting from injury and causes inflammation and apoptosis, observed normally in development and now identified as programmed cell death. Apoptosis and necrosis are types of cell death. They are generally considered to be distinct forms of cell death, but there is mounting evidence supporting an apoptosisnecrosis cell death continuum (Portera-Calliau, et al, 1997). In this continuum, neuronal death can result from varying contributions of coexisting apoptotic and necrotic mechanisms, resulting in some of the distinctions between apoptosis and necrosis becoming blurred. Today it is believed that apoptosis may contribute to the neuronal degeneration in neurological injuries such as cerebral ischemia and trauma (Kerr et al

Necrosis can result from acute oxidative stress characterized by passive cell swelling, rapid energy losss, and generalized disruption of internal homeostasis with lysis of the nucleus, intra-nuclear organelles and plasma membranes leading to the release of intracellular components that induce a local inflammatory response that in turn, result in edema and injury to neighboring cells. Morphologically, cell death is characterized by swelling of organelles and rupture. Necrotic cell death is characterized by inflammation and wide-

Apoptosis is a process of cell suicide, the mechanisms of which are encoded in the chromosomes of all nucleated cells. Although the capacity to carry out apoptosis appears to be inherent in all cells, the susceptibility to apoptosis varies markedly and is influenced by external and cell-autonomous events. Apoptosis is regulated by complex molecular signaling systems resulting in an orderly, energy-dependent enzymatic breakdown into characteristic molecular fragments, DNA, lipids and other macromolecules. Apoptosis can be induced by cell surface receptor engagement, growth factor withdrawal and DNA damage. In contrast to those observed in cell necrosis, the morphological changes that occur during developmental cell death include cell shrinkage, membrane blebbing, chromatin condensation and DNA fragmentation. Earlier studies showed that one of the biochemical hallmarks of apoptosis is DNA cleavage at internucleosomal linker regions, resulting in ladder formation of DNA of 180-200 bp or multiples thereof. However, this ladder-type DNA fragmentation is also found in some cells dying of necrosis, indicating that DNA fragmentation cannot be the sole criterion, but simultaneous morphological assessment

Several families of proteins and specific biochemical signal-transduction pathways regulate cell death. Cell death signaling can involve plasma membrane death receptors, mitochondrial death proteins, proteases, kinases and transcription factors. Predominant factors in cell death and cell survival include fas receptor, Bcl-2 and Bax (and their homologues), cytochrome c, caspases, p53 and extra cellular signal-regulated protein kinases. Some forms of cell death require gene activation, RNA synthesis and protein synthesis, whereas other forms are transcriptionally-translationally independent and are

**8. Neuronal injury and nerve cell death: basic sciences** 

1972; MacManus and Linnik, 1997; Martin, 2001).

must also be done for identifying apoptosis.

**8.1 Mechanisms of apoptotic cell death** 

spread damage.

or 10 oC HCA, respectively. Systemic pressure was maintained above 60 mmHg during reperfusion. Measurements of hemodynamics (heart rate, mean arterial pressure), arterial blood gases, hematocrit, glucose lactate, as well as temperatures were recorded at five time points during the experiment. These were: 1) Baseline at 37oC and prior to CPB; 2) At the initiation of CPB; 3) During CPB, while cooling to a brain temperature of 18oC or 10oC just before HCA; 4) During rewarming; and 5) At the end of CPB.

The mean duration (+SD) of CPB cooling and CPB warming for animals with 18oC HCA was 57.50+17.25 and 82.50+10.37 minutes, and for 10oC was 105.8±21.8 and 104.2±19.8 minutes, respectively. Perioperative physiological variables are shown in Table 1. Although there were some minor variations, no apparent clinically relevant hemodynamic differences were observed between treatment groups. Lactate levels were significantly higher following HCA at 10oC compared to 18oC. PO2 levels were significantly lower in 18oC HCA animals compared to 10oC during cooling, and hematocrit levels dropped to a similar degree in all experimental animals during the procedure.


Table 1. Typical Physiologic Variables During HCA Paradigm in a Porcine Model All values are expressed as mean + SD. **\***P<0.05 between animals treated with HCA at 18oC vs 10oC (unpaired, 2-tailed, t-test). **\*\***P<0.05 between sample times (ANOVA followed by Fisher PLSD).

or 10 oC HCA, respectively. Systemic pressure was maintained above 60 mmHg during reperfusion. Measurements of hemodynamics (heart rate, mean arterial pressure), arterial blood gases, hematocrit, glucose lactate, as well as temperatures were recorded at five time points during the experiment. These were: 1) Baseline at 37oC and prior to CPB; 2) At the initiation of CPB; 3) During CPB, while cooling to a brain temperature of 18oC or 10oC just

The mean duration (+SD) of CPB cooling and CPB warming for animals with 18oC HCA was 57.50+17.25 and 82.50+10.37 minutes, and for 10oC was 105.8±21.8 and 104.2±19.8 minutes, respectively. Perioperative physiological variables are shown in Table 1. Although there were some minor variations, no apparent clinically relevant hemodynamic differences were observed between treatment groups. Lactate levels were significantly higher following HCA at 10oC compared to 18oC. PO2 levels were significantly lower in 18oC HCA animals compared to 10oC during cooling, and hematocrit levels dropped to a similar degree in all

**Variable Baseline Init CPB Cooling Warming End CPB** 

**18 oC** 36.5+0.4 34.1+1.8 18.0+0.0 25.8+3.2 36.5+0.8 **10 oC** 36.5+0.4 33.2+1.6 10.0+0.0 28.2+3.1 36.9+0.2

**18 oC** 114.0+14.9 57.2+16.3 55.2+8.1 67.8+15.7 68.3+25.7 **10 oC** 118.7+13.0 59.7+10.1 54.0+3.4 69.4+16.5 85.0+8.9

**18 oC** 7.40+0.12 7.26+0.19 7.26+0.12 7.20+0.07 7.35+0.14 **10 oC** 7.34+0.13 7.32+0.11 7.28+0.11 7.32+0.08 7.38+0.13

**18 oC** 409.9+67.8 751.4+202.8 787.1+319.06**\*** 429.0+126.9 424.6+112.1 **10 oC** 378.4+118.3 689.5+45.5 1066.0+122.8 562.8+123.4 459.4+45.4

**18 oC** 51.32+19.0 73.7+37.8 67.2+31.3 69.3+14.4 38.7+18.7 **10 oC** 58.1+23.3 60.0+17.7 58.2+12.4 44.0+12.9 31.7+9.0

**18 oC** 26.4+3.8**\*\*** 16.4+3.4 15.51+3.3 16.6+3.9 15.5+3.1 **10 oC** 26.0+3.8**\*\*** 18.5+3.0 18.6+3.7 19.2+3.5 19.2+3.0

**18 oC** 2.7+1.8 4.5+2.1 5.6+2.5 6.3+1.7**\*** 11.0+4.3 **10 oC** 3.1+1.03 4.4+1.6 8.3+3.0 11.6+2.8 11.9+3.5

All values are expressed as mean + SD. **\***P<0.05 between animals treated with HCA at 18oC vs 10oC (unpaired, 2-tailed, t-test). **\*\***P<0.05 between sample times (ANOVA followed by

Table 1. Typical Physiologic Variables During HCA Paradigm in a Porcine Model

before HCA; 4) During rewarming; and 5) At the end of CPB.

experimental animals during the procedure.

**Brain Temperature (oC)**

**MAP (mmHg)**

**Arterial pH** 

**pO2 (mmHg)**

**pCO2 (mmHg)**

**Hematocrit (%)**

**Lactate (mmol/L)**

Fisher PLSD).
