Differential Cerebral Gene Expression During Cardiopulmonary Bypass in the Rat: Evidence for Apoptosis? Yukie Sato, MD*, Daniel T. Laskowitz, MD†, Ellen R. Bennett, PhD‡, Mark F. Newman, MD*, David S. Warner, MD*, and Hilary P. Grocott, MD, FRCPC* Departments of *Anesthesiology, †Medicine (Neurology), and ‡Pathology, Duke University Medical Center, Durham, North Carolina Cardiopulmonary bypass (CPB) is associated with a spec- trum of cerebral injuries. The molecular changes in the brain that might contribute to these injuries are not clearly known. We sought to determine whether the expression of apoptotic genes is increased after CPB in the rat. Rats (n = 7) were subjected to 90 min of normothermic CPB. A group of sham-operated rats (n = 7) served as non-CPB controls. After a 3-h post-CPB period of recovery, their brains were removed, homogenized, and processed for messenger RNA (mRNA) extraction. By using a ribonu- clease protection assay, the ratios of both pro- and anti- apoptotic mRNA (bcl-x, bcl-2, bax, caspase 2, and caspase 3) to the housekeeping glyceraldehyde phosphate dehy- drogenase (GAPDH) gene were determined. Addition- ally, Western immunoblotting was performed to detect the presence of activated caspase 3, a protein central in the apoptotic process. Compared with the non-CPB controls, the CPB group had significantly increased levels of apoptotic/GAPDH mRNA ratios (bcl-x, 0.414  0.152 CPB versus 0.251  0.051 non-CPB, P = 0.048; caspase 2, 0.030  0.014 CPB versus 0.018  0.005 non-CPB, P = 0.048; bax, 0.106  0.035 CPB versus 0.066  0.009 non- CPB, P = 0.009; bcl-2, 0.011  0.006 CPB versus 0.006  0.002 non-CPB, P = 0.035). However, no activated caspase 3 protein was detected in either group. Elucidating the molecular biological sequelae of CPB may aid in the un- derstanding of the pathophysiology of cardiac surgery- associated cerebral injury and, in doing so, may be useful in identifying potential therapeutic targets for pharmaco- logic neuroprotection. (Anesth Analg 2002;94:1389 –94) A dverse cerebral outcomes after cardiopulmo- nary bypass (CPB) for cardiac surgery have been well documented (1–7). These injuries en- compass a complete spectrum, from subtle cognitive impairment to overt stroke. The etiology of these ce- rebral injuries, although not clearly understood, prob- ably represents a complex interaction among cerebral microemboli (8,9), global cerebral hypoperfusion (10), inflammation (11), cerebral temperature modulation (12–15), and genetic susceptibility (16). Although the cerebral consequences of CPB have been measured clinically, insights into the molecular events within the brain occurring as a result of CPB have only begun to be investigated (17,18). Apoptosis is a well documented series of events that results in the programmed self-destruction of cells. Its stimulus for initiation is variable but includes ische- mia and other stresses (19,20). These stresses induce an intracellular molecular cascade that ultimately re- sults in self-destruction of tissue. Apoptosis is respon- sible for the long-term loss of neuronal tissue after cerebral ischemia (21), but its potential role in CPB- associated cerebral injury is not clearly known. Gaining insights into the cerebral consequences of CPB, and in particular the molecular pathways possibly involved, has been limited by the relative inability to study brain tissue after recovery from CPB. With the development of a rat model of CPB (22,23), we have been able to sample brain tissue after CPB for molecular anal- ysis with well established molecular tools. In this study, we hypothesized that the transcription of apoptotic genes would be increased after CPB and that protein evidence for apoptosis would confirm the initiation of this self-destructive cellular process. Methods After Duke University Animal Care and Use Commit- tee approval, the following experiment (adhering to Dr. Grocott was supported by a Pepper Center Junior Faculty Award (NIA-AG11268). Accepted for publication February 13, 2002. Address correspondence and reprint requests to Hilary Grocott, MD, Associate Professor of Anesthesiology, Department of Anes- thesiology, Duke University Medical Center, Box 3094, Durham, NC 27710. Address e-mail to h.grocott@duke.edu. ©2002 by the International Anesthesia Research Society 0003-2999/02 Anesth Analg 2002;94:1389–94 1389