This study examined canine subjects that underwent 2-hour HCA, 1-hour HCA, or CPB alone to determine the gene expression profiles within a particularly vulnerable area of the brain (hippocampus). The data show greater numbers of genes regulated after longer-duration HCA, which also produced clinically worse neurologic impairment. For example, dogs undergoing 2-hour HCA had increased numbers of genes regulated compared with 1-hour HCA, whereas dogs undergoing CPB alone had either no alterations in gene expression (24 hours) or minimal numbers of regulated genes (5 at 8 hours). This is notable given the high sensitivity of the relatively low absolute fold change significance requirement (≥1.2), the purpose of which was to cast a wide net for potentially regulated genes. These changes were consistent with the severity of clinical neurologic injury in each group. The lack of significant gene regulation in CPB dogs is surprising, given this highly sensitive genomic analysis. This finding supports the contention that standard CPB has negligible long-term effects on a normal brain. This is consistent with our previous work investigating brain injury biomarkers after CPB and HCA, in which no change in levels of spectrin breakdown products were observed in CPB dogs, whereas there was a graded increase with longer durations of HCA [16
Our 1-hour HCA model is meant to represent clinically relevant, yet prolonged, HCA duration. The 2-hour HCA model is purely experimental. It achieves a consistent severe neurologic injury (on pathologic examination, neurologic scoring, biomarker analysis, and genomic analysis), and is used to investigate potential therapeutics and compare findings in 1-hour HCA dogs. Examining the pattern of gene expression, 2-hour HCA dogs had more gene regulation at 24 hours than at 8 hours after injury, suggesting progression of injury with time. Conversely, in 1-hour HCA dogs, many genes regulated at 8 hours had returned to baseline by 24 hours (). Coupled with the overlap in regulated genes in the two HCA groups, a pattern of gene regulation in HCA brain injury emerges, with proapoptotic genes regulated early and antiapoptotic and immune response genes regulated later (24 hours).
Fig 5 Summary plot of the temporal pattern of gene expression in each treatment group with notable overlapping gene ontology categories regulated at each time. (CPB = cardiopulmonary bypass; 1H HCA = 1 hour of hypothermic circulatory arrest; 2H HCA = 2 hours (more ...)
Examining the biologic process categories regulated in 1-hour HCA dogs yields insight into the cellular responses that are initiated in the brain after a clinically relevant duration of HCA. At 8 hours, the regulated genes are clustered in areas such as apoptosis, inflammatory response, defense response, response to wounding, and developmental processes. At 24 hours, fewer apoptotic and developmental genes are regulated. Additionally, there is increased activity in other immune system mechanisms, such as complement activation and cell motion. However, inflammatory and defense responses remain significantly regulated at both 8 and 24 hours after HCA. Although 10% to 20% of regulated genes in each group were regulated at both times, further elucidation of important pathways is necessary to identify their significance.
It appears that the brain’s response to HCA in the first 24 hours is marked by early programed cell death and initiation of developmental processes, late immune system and complement activation, and regulation of the inflammatory and defensive responses. These findings are interesting when taken in the context of specific genes that are regulated and have been previously reported to influence brain injury after HCA.
Previous animal studies have reported the hippocampus to be particularly vulnerable to injury after HCA [8
]. Research in animal models and patients have suggested a regulation of inflammatory and apoptotic genes after CPB and HCA [24
]. However, our study reports the transcriptional profiling of animals undergoing HCA and CPB. Microassays have been previously used to study changes in gene expression after HCA and CPB in rodents [25
]. That genes of known importance in the pathway of glutamate excitotoxicity, mitochondrial injury, and early apoptosis are regulated in our study helps to support and extend our previous investigations on the importance of this pathway for the post-HCA brain [27
Two studies of interest involve the expression of apoptotic genes in models of HCA and CPB. Ananiadou and colleagues [24
] demonstrated increased levels of the anti-apoptotic mitochondrial protein BCL2
within porcine hippocampus 3 hours after 75 minutes of 18°C HCA. This is consistent with our observations that apoptotic genes are significantly regulated after clinically relevant HCA. Zhang and colleagues [28
] also implicated regional increases in apoptotic mitochondrial proteins BCL2
in the hippocampus up to 6 hours after hypothermic CPB in a rodent model. Among the five BCL2 family members examined in our study, BCL2A1 was most highly regulated; with fourfold to fivefold increased expression at both times after 2-hour HCA and at 8 hours after 1-hour HCA.
This study builds on previous work on brain injury after HCA and CPB from our laboratory and others by applying the emerging technologies of canine genomics in our established model. This transcriptional profiling of the 24 hours after these procedures affirms the importance of inflammatory and apoptotic processes in the response of the brain to HCA. This characterization of transcriptional regulation holds promise to identify potential therapeutic targets and greater mechanistic understanding as the specifics of the genes involved and their interactions are more fully elucidated.
Our study is limited by our small sample size and limited information on the temporal pattern of gene expression. Our goal was to focus on short-term changes in gene expression and to begin defining the temporal relationships of expression in different biologic process GO terms. We acknowledge the current study provides little information on long-term changes in gene expression. Additionally, we recognize that the effects of CPB and HCA in comorbid adult patients undergoing cardiac surgery could differ from those in young and healthy canine subjects. However, the molecular mechanisms and gene regulation after HCA-induced injury are likely similar. Our experimental design is further limited by lack of control groups receiving 18°C CBP alone (without HCA) or anesthesia alone. Hence, we cannot comment on the effect of arrest beyond hypothermia on brain injury or the impact of anesthesia alone on gene expression. As well, our 1-hour HCA duration is longer than routine clinical HCA, but represents the upper limit of acceptable clinical HCA duration.
Brain injury after cardiac surgery remains a significant clinical concern, the origin of which is not completely understood. Genomics provides a window into the transcriptional profile of the brain after HCA and CPB. In this initial analysis, CPB leads to minimal changes in gene regulation, whereas HCA results in significant regulation of several genes, including those affecting apoptosis and inflammation. The magnitude of these alterations in gene expression increases with duration of HCA.