**8.4 The decision phase for apoptotic nerve cell death: evidence for Bcl-2**

Apoptosis is controlled genetically, and two genes, Bcl-2 and p53 are now believed to be important. It is now established that proteins encoded by the Bcl-2 gene family are major regulatory components of the apoptotic pathway (Kroemer, 1997). Within the apoptotic cascade, several proteins that facilitate neuronal survival compete with molecules that contribute to cell death. Ultimately, the final balance between cell survival–promoting proteins versus cell death–promoting proteins determines the fate of the cell. The Bcl-2 family of proteins plays an important role in this cell survival-cell death decision.

This hypothesis is supported by our findings in Bcl-2 expression. The Bcl-2 family of proteins is important for the regulation of apoptosis during the "decision phase." An increase of Bcl-2 has been suggested as an internal protective mechanism against apoptotic cell death, where Bcl-2 is persistently expressed in neurons that survive in ischemia. In the present study, brain regions that were selectively vulnerable to neurologic injury, particularly the neocortex and hippocampus, showed higher levels of Bcl-2 expression after HCA at 18°C compared with other brain regions (thalamus, cerebellum, and medulla). Moreover, profound hypothermia at 10°C resulted in a significant decrease in TUNEL staining in these brain regions. Although a concomitant increase in Bcl-2 expression was observed in the neocortex, it remains unclear whether profound hypothermia deters from neuronal injury by activation of anti-apoptotic protein Bcl-2 expression (Ananiadou et al, 2007). (Table 3)


Table 3. TUNEL Scores and Bcl-2 Immunoreactivity in the Sensory Neocortex of Animals Treated with HCA at 18oC or 10oC Compared to Controls

All values are expressed as mean + SE.

\*p<0.05 compared to values from animals treated with 18oC HCA.

\*\*p<0.0001 compared to values from animals treated with 10oC HCA.

\*\*\*p<0.002 compared to normal control levels

Neurologic Injury Following Hypothermic Circulatory Arrest 257

thin astrocytic end feet and showed endothelial cells with euchromatic nuclei, tight

Compared to normal controls, treatment with HCA at 18°C (group A) resulted in minor ultrastructural alterations in the sensory cortex. (Table 4) Most neurons exhibited a pale round or oval nuclei. Nucleoli were intact, although sometimes they were localized in eccentric positions. Plasma and nuclear membranes remained intact, as did the mitochondria, which maintained their normal appearance, with recognizable cristae. While most neurons had slightly dilated RER and Golgi apparatus, some cells displayed more significant edema and morphological modifications of their organelles and dilated mitochondria. (Figure 3). In some cases, polysomes were disassociated, displaying desegregated ribosomes. In addition, some neurons, also exhibited some chromatin clumps. Although some mitochondria were slightly

Sensory cortex from HCA at 18oC. (Left) Electron micrograph following HCA at 18oC with detail of neuron, showing nucleus(x18400). (Right) The same neuron showed dilated rough

Sensory cortex

*Chromatin Dispersion*  10oC 18oC

1 - - - - - - - - - + - + 2 - - - - - - - - - - - - 3 - - - - - - - - - + - + 4 - - - - - - - - - + - + Table 4. Ultrastructural Changes in the Sensory Neocortex of Animals Treated with HCA at

neuron Nuclear changes Cell organelles

*Clumping* 

*Mitochondri al Swelling*  10oC 18oC

*RER Swelling*  10oC 18oC

10oC 18oC

junctions between the adjacent plasma membranes.

dilated, they showed an otherwise normal morphology.

Fig. 3. Ultrastructural Changes in the Sensory Cortex

Overall shape of the

*Swollen* 

10oC 18oC

endoplasmic reticulum (x25200).

*Shrunken* 

10oC 18oC

18oC or 10oC Compared to Controls - No changes noted compared to control

+ Positive observation compare to control animals

Treatment

#### **8.5 Morphological and ultrastructural evidence of neural protectin during profound cooling**

Necrosis can be characterized by passive cell swelling, rapid energy losss, and generalized disruption of internal homeostasis with lysis of the nucleus, intranuclear organelles and plasma membranes leading to the release of intracellualar 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 characterised by inflammation and wide-spread damage. In contrast to those observed in cell necrosis, the morphological changes that occur during apoptotic cell death include cell shrinkage, membrane blebbing, chromatin condensation and DNA fragmentaion (Kerr et al., 1972). In continuation of the above studies, we assessed the morphological evidence that profound cooling of the cortex to 10°C can reduce neurological injury during hypothermic circulatory arrest (HCA) in our porcine model. Electron microscopy assessed ultrastructural changes indicative of activation of programmed cell death.

Parafin embedded samples described above were dewaxed in xylene. After rehydration using graded ethanol, slices were washed in cold 0.1 M sodium cacodylate buffer and fixed in 2.5% glutaraldeyde in 0.1 M cacodylate buffer overnight. Samples were washed in cold cacodylate buffer and then post-fixed with 1% osmium tetroxide in the same buffer for 1 hr at room temperature. After osmium tetroxide treatment, the samples were washed with 0.1 M sodium cacodylate buffer. The selected area was identified with a dissecting microscope, and 2x2x2 mm sections were cut out from the coronal slices and dehydrated in a graded series of ethanol, before being embedded in epoxy resin. Blocks were trimmed, and semithin 0.5-μm sections were cut with an ultramicrotome and stained with toluidine blue for light microscopy analysis. Ultrathin (75-90 mm) sections were cut and placed on 200-mesh copper grids for double-staining with uranyl acetate and lead citrate. Samples were examined with a JEOL JEM 100 CX-II electron microscope.

Samples were examined in a blind fashion by one observer using a JEOL 100CX electron microscope who was instructed to find 10 representative neurons (per experimental brain specimen) as identified by a typical nucleus and surrounding perikaryon. Two blinded investigators using an objective grading system analyzed electron micrographs of these neurons. Each investigator was asked to examine each neuron for evidence of nuclear changes (chromatin dispersion or clumping), for the presence of cytoplasmic changes, for the overall shape of the neuron (shrunken, swollen) and the appearance of rough endoplasmic reticulum (RER) compared with matched controls. Similarly, each investigator was instructed to examine the perinuclear neuronal mitochondria for abnormalities in mitochondrial distribution or shape, matrix density, crystal structure, and appearance of any abnormal structures compared with matched controls; each finding was indicated as mild, moderate, or severe depending on its frequency.

Electron microscopic observations in our study provided no appreciable morphological evidence to confirm apoptosis or necrosis of the sensory cortical neurons in this acute paradigm of HCA. In general, neurons showed normal nuclear and cytoplasmic morphology in all three treatment groups, with only minor ultrastructural changes observed ater HCA at 18°C. There was no evidence of cells swelling. The neurons of the sensory cortex in control animals had large round or oval nuclei with an evenly distributed chromatin. No discontinuities were found in cytoplasmic membrane and nucleolemma. Well-developed rough endoplasmic reticulum (RER) that was arranged in parallel stacks was observed in the cytoplasm. Polyribosomes formed characteristic rosettes, and mitochondria appeared normal in all control animals. Blood capillaries were surrounded by

Necrosis can be characterized by passive cell swelling, rapid energy losss, and generalized disruption of internal homeostasis with lysis of the nucleus, intranuclear organelles and plasma membranes leading to the release of intracellualar 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 characterised by inflammation and wide-spread damage. In contrast to those observed in cell necrosis, the morphological changes that occur during apoptotic cell death include cell shrinkage, membrane blebbing, chromatin condensation and DNA fragmentaion (Kerr et al., 1972). In continuation of the above studies, we assessed the morphological evidence that profound cooling of the cortex to 10°C can reduce neurological injury during hypothermic circulatory arrest (HCA) in our porcine model. Electron microscopy assessed ultrastructural changes indicative of activation of programmed cell

Parafin embedded samples described above were dewaxed in xylene. After rehydration using graded ethanol, slices were washed in cold 0.1 M sodium cacodylate buffer and fixed in 2.5% glutaraldeyde in 0.1 M cacodylate buffer overnight. Samples were washed in cold cacodylate buffer and then post-fixed with 1% osmium tetroxide in the same buffer for 1 hr at room temperature. After osmium tetroxide treatment, the samples were washed with 0.1 M sodium cacodylate buffer. The selected area was identified with a dissecting microscope, and 2x2x2 mm sections were cut out from the coronal slices and dehydrated in a graded series of ethanol, before being embedded in epoxy resin. Blocks were trimmed, and semithin 0.5-μm sections were cut with an ultramicrotome and stained with toluidine blue for light microscopy analysis. Ultrathin (75-90 mm) sections were cut and placed on 200-mesh copper grids for double-staining with uranyl acetate and lead citrate. Samples were examined with

Samples were examined in a blind fashion by one observer using a JEOL 100CX electron microscope who was instructed to find 10 representative neurons (per experimental brain specimen) as identified by a typical nucleus and surrounding perikaryon. Two blinded investigators using an objective grading system analyzed electron micrographs of these neurons. Each investigator was asked to examine each neuron for evidence of nuclear changes (chromatin dispersion or clumping), for the presence of cytoplasmic changes, for the overall shape of the neuron (shrunken, swollen) and the appearance of rough endoplasmic reticulum (RER) compared with matched controls. Similarly, each investigator was instructed to examine the perinuclear neuronal mitochondria for abnormalities in mitochondrial distribution or shape, matrix density, crystal structure, and appearance of any abnormal structures compared with matched controls; each finding was indicated as

Electron microscopic observations in our study provided no appreciable morphological evidence to confirm apoptosis or necrosis of the sensory cortical neurons in this acute paradigm of HCA. In general, neurons showed normal nuclear and cytoplasmic morphology in all three treatment groups, with only minor ultrastructural changes observed ater HCA at 18°C. There was no evidence of cells swelling. The neurons of the sensory cortex in control animals had large round or oval nuclei with an evenly distributed chromatin. No discontinuities were found in cytoplasmic membrane and nucleolemma. Well-developed rough endoplasmic reticulum (RER) that was arranged in parallel stacks was observed in the cytoplasm. Polyribosomes formed characteristic rosettes, and mitochondria appeared normal in all control animals. Blood capillaries were surrounded by

**8.5 Morphological and ultrastructural evidence of neural protectin during profound** 

**cooling** 

death.

a JEOL JEM 100 CX-II electron microscope.

mild, moderate, or severe depending on its frequency.

thin astrocytic end feet and showed endothelial cells with euchromatic nuclei, tight junctions between the adjacent plasma membranes.

Compared to normal controls, treatment with HCA at 18°C (group A) resulted in minor ultrastructural alterations in the sensory cortex. (Table 4) Most neurons exhibited a pale round or oval nuclei. Nucleoli were intact, although sometimes they were localized in eccentric positions. Plasma and nuclear membranes remained intact, as did the mitochondria, which maintained their normal appearance, with recognizable cristae. While most neurons had slightly dilated RER and Golgi apparatus, some cells displayed more significant edema and morphological modifications of their organelles and dilated mitochondria. (Figure 3). In some cases, polysomes were disassociated, displaying desegregated ribosomes. In addition, some neurons, also exhibited some chromatin clumps. Although some mitochondria were slightly dilated, they showed an otherwise normal morphology.

Fig. 3. Ultrastructural Changes in the Sensory Cortex Sensory cortex from HCA at 18oC. (Left) Electron micrograph following HCA at 18oC with detail of neuron, showing nucleus(x18400). (Right) The same neuron showed dilated rough endoplasmic reticulum (x25200).


Table 4. Ultrastructural Changes in the Sensory Neocortex of Animals Treated with HCA at 18oC or 10oC Compared to Controls


+ Positive observation compare to control animals

Neurologic Injury Following Hypothermic Circulatory Arrest 259

organ with a functional anatomy that is difficult both to understand and assess. Experimental and clinical studies have shown that the mechanism of neural injury is multifactorial. As such, discussions regarding the best surgical strategies for neuroprotection during circulatory arrest are formidable, at best. Although we are armed with excellent experimental and clinical studies that demonstrate the deleterious effects of prolonged exposure to cardiopulmonary bypass (CPB) on brain function and structure, the various neuroprotective strategies, particularly that of deep hypothermic circulatory arrest (DHCA) remain an issue of debate. This is related in part to the gap between the basic science understanding of brain injury caused by these events and the clinical application of

Our goal has been to assess a possible mechanism of the neuronal injury (eg apoptosis) following DHCA. As this appears to involve a subtle and complex cascade of events, we decided to apply a paradigm that on the one hand may not be totally clinically relevant, but on the other hand would allow a robust response for assessment. Further study is clearly warranted to unravel relevant mechanisms and sensitive markers, which in turn, would allow us to appreciate the potential clinical relevance of these experimental findings. 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

various neuroprotective strategies and their subsequent clinical outcomes.

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

intervention.

rewarming, pH management, among others.

There is now convincing evidence that there is a general relationship between CNS damage and increasing duration of DHCA. Although one of the goals of experimental studies is to assess the upper safe limit of DHCA, in order to do so we must more clearly understand the mechanism of cerebral injury. In most animal models, an extended period of arrest is necessary to produce a consistent and reproducible level of neuronal injury that would facilitate elucidating the mechanisms of injury, as well as the efficacy of potential neuroprotective interventions. Many large animal models require DHCA for at least 90-120 minutes in order 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

Profound hypothermia of the brain results in a reduction of cerebral blood flow and steady state cerebral oxygen consumption (considered a true index of brain metabolic activity). Research in laboratory animals and clinical observations have now documented that considerable residual cerebral metabolism remains with cooling to levels of 15-18oC, particularly when cooling interval are short. Both experimental and clinical paradigms are faced with unresolved issues, including cooling gradients, nonuniformity of brain cooling,

Various strategies have been addressed in an effort to reduce neurological complications, including profound hypothermia, antegrade cerebral perfusion, retrograde cerebral

Deep hypothermia at 10°C resulted in negligent ultrastructural changes in the sensory cortex. Most neurons exhibited pale round or oval nuclei and intact nucleoli. In a few cases, the nucleoli were localized in eccentric positions. Plasma and nuclear membranes remained intact, and the structure of the cytoplasmic organelles was similar to that observed in control animals.

The first stage or the decision phase of apoptosis is initiated after an appropriate stimulus. This is referred to as the genetic control point of cell death and appears to be regulated by the Bcl-2 family of genes. The "execution phase" which follows is responsible for the morphologic changes of apoptosis (Kam and Ferch, 2000). The absence of clear morphologic evidence of apoptosis potentially suggests that these observations may represent an early point of activation of the apoptotic pathway (decision phase), which is supported by the Bcl-2 expression findings. As important regulators of the "decision phase," an increase of Bcl-2 may represent an internal protective mechanism against apoptotic cell death. Bcl-2 is persistently expressed in neurons that survive in ischemia. The sensory cortex is selectively vulnerable to neurologic injury and showed high levels of Bcl-2 expression after HCA at 18°C compared (Ananiadou et al., 2008). Moreover, profound hypothermia at 10°C resulted in a significant decrease in TUNEL(+) staining in this brain region. The concomitant increase in Bcl-2 expression that was observed in the sensory neocortex, suggests that profound hypothermia (at 10°C) may deter from neuronal injury by activation of anti-apoptotic protein Bcl-2 expression (Almeida et al., 2000). Although subtle, electron microscopy showed that at 18°C cells exhibited dilation of the rough endoplasmic reticulum and mitochondria, ribosome detachment and Golgi derived vacuolation, while at 10°C cells exhibited only dilation of the rough endoplasmic reticulum and ribosome detachment, indicating Phase II and Phase I of the apoptotic process, respectively.

The observation that TUNEL labeled cells may eventually, but not necessarily, progress into morphologically distinct apoptotic cells also confirms the idea that different morphologic characteristics may reflect different stages of the same death process. This is supported by our electron microscopy findings. At 18°C HCA, the neurons of the sensory cortex displayed dilation of the rough endoplasmic reticulum and detachment of ribosomes, along with Golgi derived vacuolation. According to Portera-Cailliau and colleagues (Portera-Calliau, et al., 1997), these findings suggest that the sensory cortex was in Phase II of the apoptotic process. At 10°C hypothermia, the ultrastructural findings indicate that the sensory cortex was in Phase I, showing only dilation of the rough endoplasmic reticulum and detachment of ribosomes. Although both groups appear to be in earlier stages of apoptosis, the findings clearly indicate that HCA at 18°C is associated with more morphological characteristics of apoptosis, compared to 10°C.

Although subtle, electron microscopy showed that at 18°C cells exhibited dilation of the rough endoplasmic reticulum and mitochondria, ribosome detachment and Golgi derived vacuolation, while at 10°C cells exhibited only slight changes. Our findings of significantly reduced TUNEL(+) staining, a concomitant increase in Bcl-2 expression and slightly decreased ultrastructural evidence of activation of programmed cell death support that deep hypothermia at 10°C further protects the sensory neocortex.

## **9. Conclusions**

Cardiac surgeons are faced with the challenge of protecting the brain during the sensitive time of interruption of normal cerebral blood flow. The brain is an exceptionally complex

Deep hypothermia at 10°C resulted in negligent ultrastructural changes in the sensory cortex. Most neurons exhibited pale round or oval nuclei and intact nucleoli. In a few cases, the nucleoli were localized in eccentric positions. Plasma and nuclear membranes remained intact, and the structure of the cytoplasmic organelles was similar to that observed in control

The first stage or the decision phase of apoptosis is initiated after an appropriate stimulus. This is referred to as the genetic control point of cell death and appears to be regulated by the Bcl-2 family of genes. The "execution phase" which follows is responsible for the morphologic changes of apoptosis (Kam and Ferch, 2000). The absence of clear morphologic evidence of apoptosis potentially suggests that these observations may represent an early point of activation of the apoptotic pathway (decision phase), which is supported by the Bcl-2 expression findings. As important regulators of the "decision phase," an increase of Bcl-2 may represent an internal protective mechanism against apoptotic cell death. Bcl-2 is persistently expressed in neurons that survive in ischemia. The sensory cortex is selectively vulnerable to neurologic injury and showed high levels of Bcl-2 expression after HCA at 18°C compared (Ananiadou et al., 2008). Moreover, profound hypothermia at 10°C resulted in a significant decrease in TUNEL(+) staining in this brain region. The concomitant increase in Bcl-2 expression that was observed in the sensory neocortex, suggests that profound hypothermia (at 10°C) may deter from neuronal injury by activation of anti-apoptotic protein Bcl-2 expression (Almeida et al., 2000). Although subtle, electron microscopy showed that at 18°C cells exhibited dilation of the rough endoplasmic reticulum and mitochondria, ribosome detachment and Golgi derived vacuolation, while at 10°C cells exhibited only dilation of the rough endoplasmic reticulum and ribosome detachment,

The observation that TUNEL labeled cells may eventually, but not necessarily, progress into morphologically distinct apoptotic cells also confirms the idea that different morphologic characteristics may reflect different stages of the same death process. This is supported by our electron microscopy findings. At 18°C HCA, the neurons of the sensory cortex displayed dilation of the rough endoplasmic reticulum and detachment of ribosomes, along with Golgi derived vacuolation. According to Portera-Cailliau and colleagues (Portera-Calliau, et al., 1997), these findings suggest that the sensory cortex was in Phase II of the apoptotic process. At 10°C hypothermia, the ultrastructural findings indicate that the sensory cortex was in Phase I, showing only dilation of the rough endoplasmic reticulum and detachment of ribosomes. Although both groups appear to be in earlier stages of apoptosis, the findings clearly indicate that HCA at 18°C is associated with more

Although subtle, electron microscopy showed that at 18°C cells exhibited dilation of the rough endoplasmic reticulum and mitochondria, ribosome detachment and Golgi derived vacuolation, while at 10°C cells exhibited only slight changes. Our findings of significantly reduced TUNEL(+) staining, a concomitant increase in Bcl-2 expression and slightly decreased ultrastructural evidence of activation of programmed cell death support that deep

Cardiac surgeons are faced with the challenge of protecting the brain during the sensitive time of interruption of normal cerebral blood flow. The brain is an exceptionally complex

indicating Phase II and Phase I of the apoptotic process, respectively.

morphological characteristics of apoptosis, compared to 10°C.

hypothermia at 10°C further protects the sensory neocortex.

**9. Conclusions** 

animals.

organ with a functional anatomy that is difficult both to understand and assess. Experimental and clinical studies have shown that the mechanism of neural injury is multifactorial. As such, discussions regarding the best surgical strategies for neuroprotection during circulatory arrest are formidable, at best. Although we are armed with excellent experimental and clinical studies that demonstrate the deleterious effects of prolonged exposure to cardiopulmonary bypass (CPB) on brain function and structure, the various neuroprotective strategies, particularly that of deep hypothermic circulatory arrest (DHCA) remain an issue of debate. This is related in part to the gap between the basic science understanding of brain injury caused by these events and the clinical application of various neuroprotective strategies and their subsequent clinical outcomes.

Our goal has been to assess a possible mechanism of the neuronal injury (eg apoptosis) following DHCA. As this appears to involve a subtle and complex cascade of events, we decided to apply a paradigm that on the one hand may not be totally clinically relevant, but on the other hand would allow a robust response for assessment. Further study is clearly warranted to unravel relevant mechanisms and sensitive markers, which in turn, would allow us to appreciate the potential clinical relevance of these experimental findings. 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.

There is now convincing evidence that there is a general relationship between CNS damage and increasing duration of DHCA. Although one of the goals of experimental studies is to assess the upper safe limit of DHCA, in order to do so we must more clearly understand the mechanism of cerebral injury. In most animal models, an extended period of arrest is necessary to produce a consistent and reproducible level of neuronal injury that would facilitate elucidating the mechanisms of injury, as well as the efficacy of potential neuroprotective interventions. Many large animal models require DHCA for at least 90-120 minutes in order 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.

Profound hypothermia of the brain results in a reduction of cerebral blood flow and steady state cerebral oxygen consumption (considered a true index of brain metabolic activity). Research in laboratory animals and clinical observations have now documented that considerable residual cerebral metabolism remains with cooling to levels of 15-18oC, particularly when cooling interval are short. Both experimental and clinical paradigms are faced with unresolved issues, including cooling gradients, nonuniformity of brain cooling, rewarming, pH management, among others.

Various strategies have been addressed in an effort to reduce neurological complications, including profound hypothermia, antegrade cerebral perfusion, retrograde cerebral

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perfusion, etc, each with their advantages and disadvantages. Cold reperfusion has shown promising results in animal studies and needs further clinical evaluation, while pharmacological interventions may offer a very promising pathway for preventing cerebral injury.

Experimental study of a neuroprotective strategy includes appropriate selection of an animal model, as well as functional indices. Of the available animal models, selection is made with respect to their relevance and feasibility of assessing the parameters of interest. The later are identified in the context of the available data. Investigations of promising neuroprotectvie methods require validation in an experimental model (validation study), use of the method in experimental settings to optimize cerebral protection during CPB and DHCA (performance study) and test its utility during routine cardiac surgery (clinical study). Despite the plethora of experimental and clinical studies, we still require a clearer understanding of the pathophysiologic consequences of HCA. This information will be pivotal in clinical decision-making, including when to initiate circulatory arrest and the appropriate interval.

Delayed cell death via apoptotic pathways 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 in animal models. These snapshots do not permit a clear delineation of the time-line of apoptosis in the course of HCA. Because it clinical role is not clear, therapies have yet to be designed for the specific purpose of inhibiting apoptosis. Both the cascade of events and identification of pharmacologic agents that can act on molecular mediators require active investigation.

Rewarming represents a critical time period, during which any additional harm to cerebral cells might induce permanent injury or even precipitate their death. How rapidly a stable energetic and biochemical homeostasis can be obtained in order to prevent the occurrence of secondary injuries remains unclear.

Optimal perfusion characteristics required to reduce neurologic morbidity remain important issues for experimental study. While there is ample evidence supporting the effectiveness of antegrade perfusion, its optimal delivery and perfusion characteristics remain unclear.

Overall, there are still many gaps in our knowledge about how to best study cerebral outcome following DHCA. The wealth of available evidence suggests that investigations require coordinated efforts by multiple research groups, pursuing systematic, multilevel research – spanning from cell cultures, to various animal model systems ranging from rodents to large animals and ultimately to clinical trials.
