Transcriptional Profile of Brain Injury in Hypothermic Circulatory Arrest and Cardiopulmonary Bypass

doi:10.1016/j.athoracsur.2010.02.051

The Annals of Thoracic Surgery

Volume 89, Issue 6, June 2010, Pages 1965-1971

Presented at the Basic Science Forum of the Fifty-sixth Annual Meeting of the Southern Thoracic Surgical Association, Marco Island, FL, Nov 4–7, 2009.

https://doi.org/10.1016/j.athoracsur.2010.02.051 Get rights and content

Background

Little is known about the molecular mechanisms of neurologic complications after hypothermic circulatory arrest (HCA) with cardiopulmonary bypass (CPB). Canine genome sequencing allows profiling of genomic changes after HCA and CPB alone. We hypothesize that gene regulation will increase with increased severity of injury.

Methods

Dogs underwent 2-hour HCA at 18°C (n = 10), 1-hour HCA (n = 8), or 2-hour CPB at 32°C alone (n = 8). In each group, half were sacrificed at 8 hours and half at 24 hours after treatment. After neurologic scoring, brains were harvested for genomic analysis. Hippocampal RNA isolates were analyzed using canine oligonucleotide expression arrays containing 42,028 probes.

Results

Consistent with prior work, dogs that underwent 2-hour HCA experienced severe neurologic injury. One hour of HCA caused intermediate clinical damage. Cardiopulmonary bypass alone yielded normal clinical scores. Cardiopulmonary bypass, 1-hour HCA, and 2-hour HCA groups historically demonstrated increasing degrees of histopathologic damage (previously published). Exploratory analysis revealed differences in significantly regulated genes (false discovery rate < 10%, absolute fold change ≥ 1.2), with increases in differential gene expression with injury severity. At 8 hours and 24 hours after insult, 2-hour HCA dogs had 502 and 1,057 genes regulated, respectively; 1-hour HCA dogs had 179 and 56 genes regulated; and CPB alone dogs had 5 and 0 genes regulated.

Conclusions

Our genomic profile of canine brains after HCA and CPB revealed 1-hour and 2-hour HCA induced markedly increased gene regulation, in contrast to the minimal effect of CPB alone. This adds to the body of neurologic literature supporting the safety of CPB alone and the minimal effect of CPB on a normal brain, while illuminating genomic results of both.

Section snippets

Animals

For all experiments, we used a clinically relevant canine model of HCA and CPB, used in our laboratory for greater than 15 years [8, 14, 15, 16]. Conditioned, heartworm-negative, 6- to 12-month-old, 30-kg male class-A dogs were used for all experiments (Marshal Bioresources, North Rose, NY). Experiments were approved by The Johns Hopkins University School of Medicine Animal Care and Use Committee and complied with the “Guide for the Care and Use of Laboratory Animals” (1996, U.S. National

Subjects

Ten dogs underwent 2-hour HCA, 8 had 1-hour HCA, and 8 had CPB alone. Half of the dogs in each group (5 2-hour HCA and 4 1-hour HCA and CPB) were sacrificed at 8 hours and half at 24 hours. Physiologic data ensuring the consistency of each technique are reported (Table 1). There were no operative or technical complications in any group.

Neurologic Scores

Using the neurologic scoring system (0 to 480), higher scores indicate worse neurologic function. Scores at 24 hours were significantly different among groups,

Comment

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

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Inflammatory profile in a canine model of hypothermic circulatory arrest
2021, Journal of Surgical Research
Citation Excerpt :
Inflammatory mechanisms that may contribute to HCA-related neurologic injury have not been fully elucidated, limiting the discovery and adoption of additional neuroprotective strategies. Previous work in our lab has demonstrated that HCA in conjunction with CPB increases neurologic injury compared to CPB alone, and that apoptotic and inflammatory gene expression and severity of injury are proportionally related to the duration of HCA.8 While that previous study was instrumental in elucidating HCA-associated gene regulation affecting apoptosis and inflammation, its focus was limited to understanding mRNA transcriptional profiles.
Hypothermic circulatory arrest (HCA) is a technique used for complex repair of the aorta, but it can be associated with neurologic morbidity. To better understand the molecular changes that underlie ischemic brain injury, we assessed gene expression and cytokine/chemokine polypeptide concentration in brain tissue and cerebrospinal fluid (CSF) of canines that underwent two hours of HCA. Materials and Methods: Adult male canines were cannulated peripherally for cardiopulmonary bypass, cooled to 18°C, and arrested for two hours. Animals were euthanized two, eight, or 24 hours post-HCA (n = 8 per group), and their brains were compared to brains from eight normal canines, using gene expression microarray analysis, cytokine assay, and histopathology.
Two to eight hours after HCA, pro-inflammatory cytokine mRNAs increased markedly, and gene expression was enriched within signaling pathways related to neuroinflammation or ischemic injury. Concentrations of pro-inflammatory cytokine polypeptides IL-6, IL-8, IL-1β, and CCL2 were very low in normal canine brain, whereas anti-inflammatory IL-10 and TGF-β1 were expressed at moderate levels. Pro-inflammatory cytokine concentrations rose robustly in cerebral tissue and CSF after HCA. IL-6 and IL-8 peaked at eight hours and declined at 24 hours, while IL-1β and CCL2 remained elevated. Concentrations of anti-inflammatory IL-10 and TGF-β1 were maintained after HCA, with a significant increase in TGF-β1 at 24 hours.
These cytokines represent potential diagnostic markers for ischemic neurologic injury that could be used to assess neurologic injury in patients undergoing HCA. The cellular mechanisms underlying this pro-inflammatory, ischemic-induced injury represent potential targets for neuroprotection in the future.
Nanotechnology Approaches to Targeting Inflammation and Excitotoxicity in a Canine Model of Hypothermic Circulatory Arrest–Induced Brain Injury
2016, Annals of Thoracic Surgery
Citation Excerpt :
Lastly, to maintain high levels of dendrimer over the most critical period (24 to 36 hours after injury), an additional bolus dose is administered 24 hours post-HCA. Our established model has been described previously [21], and therefore, only a brief description follows. Anesthesia is initiated with methohexital sodium (12 mg/kg intravenously, in divided doses) and maintained on isoflurane inhalational anesthesia (0.5% to 2.0%), intravenous fentanyl (150 to 200 μg/dose), and midazolam (2.5 mg/dose) after endotracheal intubation.
Neurocognitive dysfunction and injury remain problematic after cardiac procedures requiring hypothermic circulatory arrest (HCA). Due to poor blood-brain barrier penetrance and toxicities associated with systemic drug therapies, clinical success has been elusive. Accordingly, we explored targeted dendrimer (a nanoparticle) drug therapies in our well-established canine model of HCA to characterize the biodistribution and cellular localization of these nanoparticles in areas of known neuronal apoptosis and necrosis.
Class A, 27- to 30-kg male hounds were administered an initial intravenous bolus (10% of the total dose [200 mg]) of generation-six polyamidoamine dendrimer (6.7 nm) labeled with cyanine 5, and cardiopulmonary bypass with peripheral cannulation was initiated. After 90 minutes of HCA, 70% of the total dose was infused over a 6-hour period. The final 20% of the total dose was given 24 hours post-HCA. The brain was harvested 48 hours later (72 hours post-HCA) and analyzed for dendrimer 6-cyanine 5 biodistribution.
The dorsal hippocampus demonstrated the highest brain accumulation of dendrimer 6-cyanine 5, which closely corresponds to the distribution of apoptotic neurons evident with histologic staining and on confocal imaging. In injured brain regions, dendrimer traversed the blood-brain barrier and localized within the target cells (injured neurons and microglia).
These findings have exciting implications for the future development of novel therapeutics to mitigate neurocognitive deficits in this group of patients.
Congenital cardiac anomalies and white matter injury
2015, Trends in Neurosciences
Cardiac abnormalities are the most common birth defects. Derangement of circulatory flow affects many vital organs; without proper supply of oxygenated blood, the brain is particularly vulnerable. Although surgical interventions have greatly reduced mortality rates, patients often suffer an array of neurological deficits throughout life. Neuroimaging provides a macroscopic assessment of brain injury and has shown that white matter (WM) is at risk. Oligodendrocytes and myelinated axons have been identified as major targets of WM injury, but still little is known about how congenital heart anomalies affect the brain at the cellular level. Further integration of animal model studies and clinical research will define novel therapeutic targets and new standards of care to prevent developmental delay associated with cardiac abnormalities.
Serum levels of neuron-specific ubiquitin carboxyl-terminal esterase-L1 predict brain injury in a canine model of hypothermic circulatory arrest
2011, Journal of Thoracic and Cardiovascular Surgery
Citation Excerpt :
Canine microarray analysis was performed in a blinded fashion at the Johns Hopkins Deep Sequencing and Microarray Core Facility. Detailed methods of this protocol have been described previously.14 Gene expression profiles in samples from ventral anterior hippocampus were compared between 2h-HCA, 1h-HCA, CPB, and untreated normal dogs (each treatment group was compared with normal controls).
Ubiquitin carboxyl-terminal esterase-L1 (UCHL1) is a protein highly selectively expressed in neurons and has been linked to neurodegenerative disease in humans. We hypothesize that UCHL1 would be an effective serum biomarker for brain injury as tested in canine models of hypothermic circulatory arrest (HCA) and cardiopulmonary bypass (CPB).
Dogs were exposed to CPB (n = 14), 1 hour of HCA (1h-HCA; n = 11), or 2 hours of HCA (2h-HCA; n = 20). Cerebrospinal fluid and serum were collected at baseline, 8 hours, and 24 hours after treatment. UCHL1 levels were measured using a sandwich enzyme-linked immunosorbent assay. Neurologic function and histopathology were scored at 24 hours, and UCHL1 immunoreactivity was examined at 8 hours.
Baseline UCHL1 protein levels in cerebrospinal fluid and serum were similar for all groups. In serum, UCHL1 levels were elevated at 8 hours after treatment for 2h-HCA subjects compared with baseline values (P < .01) and also compared with CPB dogs at 8 hours (P < .01). A serum UCHL1 level above 3.9 ng/(mg total protein) at 8 hours had the best discriminatory power for predicting functional disability. In cerebrospinal fluid, UCHL1 was elevated in all groups at 8 hours after treatment compared with baseline (P < .01). However, UCHL1 levels in cerebrospinal fluid remained elevated at 24 hours only in 2h-HCA subjects (P < .01). Functional and histopathologic scores were closely correlated (Pearson coefficient, 0.66; P < .01) and were significantly worse in 2h-HCA animals.
This is the first report associating elevated serum UCHL1 with brain injury. The novel neuronal biomarker UCHL1 is increased in serum 8 hours after severe neurologic insult in 2h-HCA animals compared with CPB animals. These results support the potential for use in cardiac surgery patients and form the basis for clinical correlation in humans.
Influence of cardiopulmonary bypass on postoperative brain cognitive function of children with congenital heart disease
2020, Chinese Journal of Applied Clinical Pediatrics
MicroRNA expression in the hippocampal CA1 region under deep hypothermic circulatory arrest
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View all citing articles on Scopus

^⁎: Recipient of the 2009 Hawley H. Seiler Resident Award.

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Hawley H. Seiler Resident Award paperTranscriptional Profile of Brain Injury in Hypothermic Circulatory Arrest and Cardiopulmonary Bypass

Background

Methods

Results

Conclusions

Section snippets

Animals

Subjects

Neurologic Scores

Comment

Ann Thorac Surg

J Comput Tomogr Vasc Anesth

Ann Thorac Surg

J Am Coll Cardiol

J Thorac Cardiovasc Surg

Ann Thorac Surg

Ann Thorac Surg

J Thorac Cardiovasc Surg

Ann Thorac Surg

J Mol Cell Cardiol

J Thorac Cardiovasc Surg

Ann Thorac Surg

Society of Thoracic Surgeons National Database Executive Summary

Adverse cerebral outcomes after coronary bypass surgeryMulticenter Study of Perioperative Ischemia Research Group and the Ischemia Research and Education Foundation Investigators

N Engl J Med

Hawley H. Seiler Resident Award paper
Transcriptional Profile of Brain Injury in Hypothermic Circulatory Arrest and Cardiopulmonary Bypass