We used our clinically relevant canine model of HCA and CPB.13,14,16,24,25
Six to 12-month-old, 30-kg male class-A dogs were used (Marshal Bioresources, North Rose, NY). The Johns Hopkins Animal Care and Use Committee approved the experimental protocols, which complied with the “Guide for the Care and Use of Laboratory Animals” (1996, U.S. National Institutes of Health).
Canines were randomly exposed to 2 hours(h) of HCA (n=20), 1h-HCA (n=11), or CPB alone (n = 14) and survived to either 8h (2h-HCA, n=10; 1h-HCA, n=5; and CPB, n=8) or 24h (2h-HCA, n=10; 1h-HCA, n=6; and CPB, n=6) after treatment. CSF and serum samples were collected at baseline (prior to the surgical incision but after induction of anesthesia), at 8h, and 24h. For both baseline and subsequent CSF collection, under sedation and in a routine sterile fashion, the spinal canal is entered with a 22G needle through the cisterna magnum (at the base of the skull posteriorly). Samples are immediately frozen in a −80°C freezer. Blood samples are obtained through previously placed peripheral intravenous catheters, cold centrifuged to collect serum, and frozen at −80°C. At the conclusion of the experiment, all subjects were euthanized by exsanguination, and brains harvested for analysis.
Surgical hypothermic circulatory arrest procedure
Anesthesia was induced with methohexital sodium 9mg/kg. Animals were endotracheally intubated and maintained on inhaled isoflurane (0.5%–2%), 100% oxygen, and intravenous fentanyl (150–200 μg/dose). Tympanic membrane, esophageal, and rectal probes monitored temperatures throughout the experiment. A left femoral artery cannula was placed for arterial blood gas and hemodynamic monitoring.
Standard CPB circuits with a40-μm arterial filter (Sorin Group, Arvada, CO) were used in all experiments. Intravenous heparin (300U/kg) was administered and the right femoral artery cannulated (12F–14F), advancing the cannula into the abdominal aorta. Two separate venous cannulas (18F–20F) were advanced to the right atrium via the right femoral and external jugular veins. Vessels were cannulated by an open cutdown technique. Closed-chest CPB was initiated using pump flows of 60–80mL · kg−1 · min−1 to maintain mean arterial pressure of 60–80mmHg, and activated clotting times were maintained >500 seconds. For animals in the 1h- or 2h-HCA groups, the pump was stopped when tympanic temperatures reached 18°C (approximately 30 minutes).
Animals underwent 1h- or 2h-HCA with alpha-stat regulation of arterial blood gases (pH, 7.3–7.4; arterial partial pressure of oxygen >300mmHg and carbon dioxide 30–40mmHg). Once HCA finished, CPB was resumed, and rewarming commenced (5°C temperature gradient every 15 minutes to a core temperature of 37°C for 2h). Intravenous phenylephrine was used when necessary to maintain mean arterial pressure >75mmHg. External defibrillation was performed when temperatures were 32°C. At 37°C, animals were separated from CPB, decannulated, and reversed with protamine (3mg/kg intravenous).
Animals recovered from anesthesia while intubated, with frequent monitoring of vital signs, arterial blood gases, and urine output. Once hemodynamically and clinically stable, they were extubated and transferred to their crate for recovery. Analgesics were administered per protocol after the procedure.
Cardiopulmonary bypass only
After induction and cannulation, animals underwent 2h of CPB without HCA. Animals were cooled to 32°C, and the heart continued to beat during this operation. Animals were recovered from anesthesia as described above.
The University of Pittsburgh Canine Neurological Score was independently determined at 24h by two non-blinded study team members.26
The score includes 22 clinical questions relating to level of consciousness, respiration, cranial nerve function, reflexes, behavior, and motor and sensory function. Animals had normal neurologic function before experimentation. No additional sedation was given within 12h of neurologic assessment.
Euthanasia and tissue procurement
Animals were sedated, intubated, and anesthetized. A sternotomy was performed, 300units/kg of heparin given, and the ascending aorta cannulated (22F). Cold perfusion via the aortic cannula flushed the brain with 12L of saline solution (4°C) at 60mmHg. The right atrial appendage was transected, and venous return suctioned into a reservoir. Brains were harvested by means of a wide craniectomy.
Tissue preparation and UCHL1 Immunohistochemistry
Brains were immersion-fixed for one week in 4% paraformaldehyde, cut into 2mm coronal blocks, embedded in paraffin and sectioned at 6 μm. For immunohistochemistry, endogenous peroxidase was quenched (3% hydrogen peroxide in methanol,10 min, RT), slides were blocked (phosphate buffered saline (PBS) with 5% normal goat serum (NGS), 0.2%triton X-100, and 0.2%gelatin, 60 min, RT) and incubated in rabbit anti-UCHL1 (Novus Biologicals NB100-65827, 1:1600 in PBS with 1.5%NGS, 0.2% Triton X-100, 0.2%gelatin and 1 μl/ml sodium azide, 48 h, 4°C). The primary antibody was visualized using the avidin-biotin-peroxidase complex method (ABC-Elite, Vector Laboratories).
Hematoxylin and eosin staining was performed for blinded histologic evaluation by a single neuropathologist (J.C.T.). Eleven distinct regions of the canine brain were evaluated for the presence of apoptosis and necrosis. These regions include midfrontal cortex, superior parietal cortex, basal ganglia, hippocampus (dentate gyrus and CA regions), entorhinal cortex, amygdala, cerebellum (molecular layer, Purkinje layer, and granule layer), and brainstem. A semiquantitative scale was used to assess the degree of necrosis and apopstosis in each region. These scores were summed to obtain the neuronal cell death score, ranging from a minimum of 0 (no damage) to a maximum of 99 (extreme neuronal damage).
UCHL1 levels in CSF and serum were measured using a UCHL1 sandwich enzyme-linked immunosorbent assay (ELISA) modified from a protocol previously reported.20,21
Mouse monoclonal anti-human UCHL1 antibody and rabbit polyclonal anti-human UCHL1 antibody were made in-house against recombinant human UCHL1 full length protein and partial protein, respectively. Both were affinity purified and specificity was confirmed by immunoblotting. Reaction wells were coated with capture antibody (purified mouse monoclonal anti-human UCHL1) in 0.05M sodium bicarbonate, pH 9.6 and incubated overnight at 4°C. Plates were then washed with blocking buffer (Tris buffer saline with 0.02% Tweeen-20 (v/v);[TBST]), and further incubated for 30 minutes at ambient temperature with gentle shaking. Antigen standard (UCHL1 standard curve:0, 0.06–15 ng/mL), unknown samples (5 μL of CSF; 20 μL of serum), or assay internal control samples were incubated overnight with detection antibody (rabbit polyclonal anti-human UCHL1, 100 μL total volume). The capture antibody coated plate was then incubated with detection antibody-sample mixture for 1.5h at room temperature, and washed using an automatic plate washer (each well rinsed with 350 μL wash buffer [TBST]). The plate was then incubated with anti-rabbit-IgG-HRP (Amersham Biosciences) at room temperature for 1h and developed with Ultra-TMB ELISA substrate (Pierce# 34028) for 10 minutes. The plate was read at 450nm with a Molecular Devices Spectramax 190 spectrophotometer. The intra-assay coefficient of variance (CV)=2.1%–7.9% while interassay CV=0.9%–10.6 % within the assay dynamic range. The limit of detection (LOD) was 0.030 ng/mL; samples with undetectable levels were assigned 50% of the LOD (i.e.,0.015ng/mL). If samples yielded a signal above the quantification range, samples were diluted and re-assayed. For all serum samples, concentrations were normalized against total serum protein concentration for the same sample (ng/mg protein).
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 to normal controls). Exploratory data analysis was performed on normalized data with a false discovery rate (FDR)<0.10 considered significantly regulated.27
To evaluate gene function, Entrez Gene IDs for human orthologs were assigned, and Ingenuity Pathways Analysis software (Ingenuity Systems, Inc., http://www.ingenuity.com
, Redwood City, CA) was used to identify proteins that interact directly with UCHL1 or are part of canonical pathways that include UCHL1. For UCHL1-related genes that were significantly regulated in one or more treatment groups, fold-change vs. normal was determined in all groups.
Neurologic scores are presented as mean ± standard deviation. One-way analysis of variance (ANOVA) compared neurologic and histology scores among groups. For all subjects, the paired comparisons t-test examined differences in levels of UCHL1 from baseline to 8h after treatment. For 24h survival animals, repeated-measures ANOVA was used to account for the repeated serum samples from within the same canine subject over time. Post-hoc pairwise comparisons were conducted using the Tukey-Kramer method. Correlations between functional and histopathological scores were assessed using linear regression and Pearson’s correlation coefficient. Threshold values for UCHL1 levels that predict severe functional impairment (neuro score >100) were determined using unadjusted logistic regression and receiver operating characteristic (ROC) curves (area under the curve >0.7 considered significant). P-values <0.05 were considered significant, and analysis was performed using STATA software (v9.2, StataCorp-LP, College Station, TX).