Rat Poly-trauma Model
The study protocol was approved by the Institutional Animal Care and Use Committee at the 711th Human Performance Wing, Joint Base San Antonio-Fort Sam Houston, in compliance with all applicable Federal regulations governing the protection of animals in research. The study is reported in accordance with ARRIVE guidelines. The experiments reported herein were conducted in compliance with the Animal Welfare Act and per the principles set forth in the “Guide for Care and Use of Laboratory Animals,” Institute of Laboratory Animals Resources, National Research Council, National Academy Press, 2011.
Male, Sprague Dawley rats (290-325g) acquired from Charles River Laboratories (Frederick, MD) were acclimated to the facility for at least seven days. Subjects were pair-housed upon arrival and single-housed following the procedure on a 12-hour light/dark cycle with ad libitum access to food and water. Food and hydration support were placed on the floor of the cage following the procedure to ensure easy access during recovery. Each subject was induced with 3-3.5% isoflurane and administered a pre-operative dose of Buprenorphine HCl (0.1mg/kg) (Reckitt Benckiser Healthcare, Slough, Berkshire). Anesthesia was maintained at 0.5-2% isoflurane and rats were placed on a physiological monitoring system (Harvard Apparatus, Holliston, MA) that regulated body temperature and recorded heart rate (HR), oxygen saturation (SpO2), systolic and diastolic blood pressure, and respiration rate (RR).
The procedure timeline is depicted in Fig. 1. First, vascular access was obtained by cannulating the femoral artery and vein with heparin coated polyethylene (PE)-50 tubing (Instech Laboratories, Inc, Plymouth Meeting, PA) and a rat femoral vein catheter (Instech Laboratories, Inc, Plymouth Meeting, PA), respectively. Mean arterial pressure (MAP) was monitored with a pressure transducer (Micro-Med INC, Louisville, KY) and Blood Pressure Analyzer (Micro-Med INC, Louisville, KY) from the arterial line. The rat was moved to the prone position and a round metal disc (10mm diameter by 3mm thick) (online metals.com, Seattle, WA) was centered and secured on the subject’s head using tissue glue. A 10-minute stabilization period followed, then pre-injury vitals were recorded (Supplemental 1). Next, the electrocardiogram leads and the SpO2 sensor were removed, the catheter lines were closed, and the subject was transferred to the TBI device consistent with the Marmarou weight drop model24. The device consisted of a platform with a 1.8m long clear plastic guide tube (22.23mm outer diameter, 15.88mm inner diameter) through which a 450g brass weight fell freely. A foam bed (type E, Foam to Size, Ashland, VA) was positioned inside a plexiglass container under the guide tube. TBI was induced by dropping the weight through the guide tube to allow contact with the metal disc on the rat’s head from a height of 1.25m24. A velocity sensor (TDS 3054B, Tektronix, Beaverton, OR) was used to ensure consistent velocity of the weight near the bottom of the guide tube was achieved for each subject (5.0 ± 0.5mm/s). Following the TBI, the rodent was quickly transferred to the physiological monitoring system, the catheters were reconnected, and the ECG leads and SpO2 sensor were placed. Isoflurane was maintained at 1% for the duration of the procedure.
After obtaining a post-TBI MAP recording, the 35% volume-controlled hemorrhage was induced using a programmable syringe pump (Legato 110, KD Scientific, Holliston, MA) at a withdrawal rate of 1mL/min from the arterial catheter. The hemorrhage volume was calculated from the total estimated blood volume for rats at 64mL/kg25. The MAP was recorded immediately following the hemorrhage then monitored continuously throughout the remainder of the surgical procedure. Next, resuscitation was initiated 15 minutes after the start of hemorrhage26. Subjects were randomly assorted into groups to receive the vehicle control or T-101 with terminal end points at four, 24, 48, or 72 hours after the start of hemorrhage (n = 5/group at each time-point). The number of animals used in this study was determined based on previous reports of brain and organ injury in rat models of TBI + hemorrhage21,27. A 1mL bolus of vehicle control (saline) or T-101 (1.7mg/kg) was administered through the venous catheter using a T-101 dosage based on previous mouse stroke data28. An additional 0.5mL saline was infused at 0.1ml/min if the MAP did not reach 70mmHg within five minutes of the bolus. Therefore, the maximum resuscitation volume was 1.5mL per rat and resuscitation was completed within 10 minutes of the bolus. After resuscitation, the cannulated vessels were ligated, catheters and leads were removed, and a post-operative dose of Buprenorphine SR (1.0mg/kg) (Zoopharm, Fort Collins, CO) was administered. The animal was returned to a fresh cage for post-surgical monitoring until the endpoint was reached. Shams underwent all surgical preparation and were maintained under anesthesia for a period of time consistent with the vehicle control and T-101 groups, but they did not experience the TBI or hemorrhage. The subjects were monitored for 72 hours following the estimated time when hemorrhage would have been initiated, and then euthanized at the 72-hour time-point with final blood collection and tissue harvesting at that 72-hour time-point.
At the assigned endpoint, all animals were placed under deep isoflurane anesthesia, given a dose of Buprenorphine HCl (0.1mg/kg), and transcardial perfusion was performed as previously described29. A final blood draw was obtained immediately before the perfusion from the left ventricle and transferred to blood collection tubes with lithium heparin or ethylenediaminetetraacetic acid (EDTA) for blood chemistry or multiplexed analyte quantification, respectively. The perfusion was performed using the Leica Perfusion Two™ automated perfusion system (Leica Biosystems, Buffalo Grove, IL) according to the manufacturer’s instructions by first infusing 10% sucrose to clear the blood, followed by 10% formalin for fixation.
Histopathology of Lung, Kidney, and Liver
The lungs, kidneys, and liver were transferred to 10% buffered formalin for at least 72 hours and then embedded in paraffin (Fisherbrand Histoplast™ PE, Pittsburg, PA). They were then sectioned to 5–6µm thickness, and stained with hematoxylin and eosin (H&E) stain (Hematoxylin+, Thermo Fisher Scientific, Pittsburg, PA; Eosin-Y Alcoholic 0.25%, Stat Lab Medical Products, McKinney, TX). One section per block was imaged and scored by a board certified veterinary pathologist. Scores were assigned on a scale of 0–5 to indicate the severity or degree of histologic finding present in examined tissue: 0 (not present), 1 (minimal), 2 (mild), 3 (moderate), 4 (marked), or 5 (severe). Cells were scored by the following scale: 0: negative, 1: <10% of cells in section are affected (minimal), 2: 11–25% of cells in section are affected (mild), 3: 26–50% of cells in section are affected (moderate), 4: 50%-75% of cells in section are affected (marked), and 5: >75% of cells in section are affected (severe).
Immunohistochemistry and Diffusion Tensor Imaging
For brain analysis, the subject’s head was fixed in 10% buffered formalin for 72 hours then transferred to phosphate buffered saline (PBS) containing 0.01% sodium azide for shipment to Biospective Inc. (Montreal, Canada) for ex vivo diffusion tensor imaging (DTI) and immunohistochemistry. Scientists at Biospective Inc. remained blinded to the treatment group during data collection and analysis. Each brain specimen was evaluated by Magnetic resonance imaging (MRI, 7T Bruker BioSepc 70/30 system, Bruker Biospin, Ettlingen, Germany). Samples were placed in the scanner and warmed to 44°C ± 0.3°C. Multi-dimensional, multi-shell images were obtained in 50 directions with three shells and six b0 images. The MRI images were processed with NIGHTWING™ software (Biospective Inc, Montreal, Canada). The lowest shell was removed from analysis due to ringing artifact. Each reconstructed image was corrected for non-uniformity with the N3 algorithm30, brain masking, and linear spatial normalization with a 12-parameter affine transformation to map individual images from a native coordinate space to a reference space. Next, an unbiased, symmetric, anatomical template was generated from the b0 images31,32 and images were linearly and nonlinearly registered to this anatomical template31,33,34. Image segmentation was achieved by identifying neuroanatomical regions on the atlas. DTI parametric maps were generated with an automated method to compute mean diffusivity (MD), axial diffusivity (AD), radial diffusivity (RD), and fractional anisotropy (FA). The parametric maps were then mapped into the template space.
Following MRI, the brain tissue was prepared for immunohistochemistry. The brain specimens were extracted from the skull and processed for paraffin embedding. Blocks were sectioned coronal in orientation at a thickness of 5µm to target the anterior, middle, and posterior segments of the corpus callosum, overlying cortex, hippocampus, and fimbria. All sections were stained with cresyl violet to label the Nissl substance in neurons and immunostained for glial fibrillary acidic protein (GFAP) to label astrocytes. First, sections were de-paraffinized and rehydrated. The cresyl violet stain was prepared as a 0.1% stock of cresyl violet acetate (Sigma-Aldrich C5042) in deinonized water. Ten drops of 10% acetic acid were added to a 30mL stock solution for each round of staining. Slides were stained for three minutes, then dehydrated and mounted with Permount mounting medium. GFAP staining was performed on a Lab Vision 360 Autostainer (Fisher Scientific, Toronto, Canada) with detergent reagents from Abcam. The slides underwent epitope retrieval by incubating them in citrate buffer (pH 6.0) and heating to 120°C under high pressure for 10 minutes. Endogenous peroxidase was quenched by sequential incubations in hydrogen peroxide for five minutes. Next, the slides were incubated in Protein Block (Abcam ab156024) for five minutes followed by 60 minutes with the primary antibody (rabbit Ab anti-GFAP, Thermo Fisher RB-087-A; 1:100). The secondary antibody (donkey anti-rabbit IgG [Jackson ImmunoResearch] and Streptavidin-HRP [Abcam ab64269]) conjugate was added for tissue visualization using AEC Single Solution (Abcam ab64252) for 20 minutes. The sections were counterstained with Acid Blue 129 (Sigma-Aldrich, St. Louis, MO) and mounted with aqueous mounting medium35. The stained sections were digitalized on an Axio Scan.Z1 digital whole slide scanner (Carl Zeiss, Canada). Image processing involved manually delineating regions of interest and using the automated PERMITS™ quantification process to determine the percent positive-stained area.
Laboratory Assays
Blood plasma collected in lithium heparin tubes were used for blood chemistries. The IDEXX Catalyst One Analyzer (IDEXX, Westbrook, Maine) clips included creatinine (CREA), creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate (LAC), and lactate dehydrogenase (LDH). Blood plasma specimens collected in EDTA tubes were evaluated for inflammatory markers. Initially, a 27-plex assay was evaluated to identify detectable analytes to down-select. Next, a customized rat four-plex cytokine/chemokine kit (Millipore, Burlington, MA) on a Bio-plex® 200 Luminex system (Bio-Rad, Hercules, CA) was used for analyses according to manufacturer’s instructions based on the analytes that had detectable levels from the 27-plex assay. The custom kit included interleukin (IL)-1β, IL-6, inflammatory protein-10 (IP-10), and leptin.
Statistical Analysis
Data are plotted as mean ± standard deviation and statistics were run on GraphPad Prism 8.3 (GraphPad Software, San Diego, CA) with P < 0.05 considered to be statistically different. Vitals data were analyzed with repeated measures ANOVA. The analysis for blood chemistry, luminex, histology and brain assessment was performed on n = 5 rats/group which survived to their end-point. Log-transformed blood chemistry and inflammation data were analyzed with a one-way ANOVA comparing injury groups and the Sham group, with Bonferroni’s multiple comparison test. Histological assessment scores were analyzed for each group compared to the Sham group via the one-way ANOVA Kruskal-Wallis test with Dunnet’s multiple comparisons post hoc test. All brain analysis data were analyzed using a one-way ANOVA and Bonferroni’s multiple comparison test. Voxel-wise statistical analysis of the DTI maps were completed using the SurfStat toolbox. The p-values without correction for multiple comparisons were reported and P < 0.05 were considered significant.