Cardiac reactive oxygen species after traumatic brain injury

Abstract

Background: Cardiovascular complications after traumatic brain injury (TBI) contribute to morbidity and mortality and may provide a target for therapy. We examined blood pressure and left ventricle contractility after TBI, and tested the hypothesis that β-adrenergic blockade would decrease oxidative stress after TBI.

Material and methods: Rodents received fluid-percussion injury or sham surgery, confirmed with magnetic resonance imaging (MRI) and histopathology. We followed recovery with sensorimotor coordination testing and blood pressure measurements. We assessed left ventricular ejection fraction using ECG-gated cardiac MRI and measured myocardial reactive oxygen species (ROS) with dihydroethidium. We randomized additional TBI and sham animals to postoperative treatment with propranolol or control, for measurement of ROS.

Results: Blood pressure and cardiac contractility were elevated 48 h after TBI. Myocardial tissue sections showed increased ROS. Treatment with propranolol diminished ROS levels following TBI.

Conclusions: TBI is associated with increased cardiac contractility and myocardial ROS; decreased myocardial ROS after β-blockade suggests that sympathetic stimulation is a mechanism of oxidative stress.

Figure 1

A. Neuroimaging with Achieva 3.0T TX magnet after TBI surgery (trauma) or sham surgery (control). Imaging obtained from animals under isofluorane anesthesia 36–48 hours following surgery. Representative sequences are shown with enhancement indicating structural injury (T2-weighted gradient and spin echo [GRaSE]), surrounding edema (fluid-attenuated inversion recovery [FLAIR]) and larger ischemic penumbra (diffusion weighted images [DWI]). B. Neurological function assessment after TBI surgery or sham surgery. Animals were pre-acclimated to the rotarod test, subjected to surgery, and allowed to recover. On the third day after surgery, animals were tested in five consecutive trials to measure length of time on the rotating rod. Values are expressed as mean +/− SEM (control, n = 17; trauma, n=16). *Different from control, two-tailed t test (P < 0.05).

Figure 1

A. Neuroimaging with Achieva 3.0T TX magnet after TBI surgery (trauma) or sham surgery (control). Imaging obtained from animals under isofluorane anesthesia 36–48 hours following surgery. Representative sequences are shown with enhancement indicating structural injury (T2-weighted gradient and spin echo [GRaSE]), surrounding edema (fluid-attenuated inversion recovery [FLAIR]) and larger ischemic penumbra (diffusion weighted images [DWI]). B. Neurological function assessment after TBI surgery or sham surgery. Animals were pre-acclimated to the rotarod test, subjected to surgery, and allowed to recover. On the third day after surgery, animals were tested in five consecutive trials to measure length of time on the rotating rod. Values are expressed as mean +/− SEM (control, n = 17; trauma, n=16). *Different from control, two-tailed t test (P < 0.05).

Figure 2

A. Immunohistochemistry for reactive gliosis after TBI surgery (trauma) or sham surgery (control). Rats were euthanized 7–10 days after surgery for acquisition of ipsilateral cortical tissue adjacent to the area of injury. Confocal microscopy was used to detect fluorescence targeted by an antibody specific to glial fibrillary acidic protein (GFAP) in frozen tissue. Representative slides are shown. B. Graph depicts GFAP fluorescence from cortical tissue sections. At least 3 microscope fields per animal were normalized to background and averaged to determine total area of fluorescence per animal. Values are expressed as mean +/− SEM (control, n = 9; trauma, n=9). *Different from control, two-tailed t test (P < 0.05).

Figure 3

A. Fluid percussion injury measured using high frequency pressure transducer in line with fluid column during procedure. The maximum pressure is shown (49 PSI) over the 500 msec time period. B. Representative tracing of systolic blood pressure after fluid percussion injury (60 second tracing). Intra-operative blood pressure was transduced from the tail artery using an indwelling line placed under anesthesia. The blood pressure increased from 100 mmHg to 153 mmHg over 3 seconds, and then returned to baseline within 30 seconds. B. Blood pressure after TBI surgery (trauma) or sham surgery (control) measured with a non-invasive tail cuff. Representative cuff pressure tracings are shown for control and trauma animals. B. Summary blood pressure data at 2 days after injury. Five to ten readings were taken and averaged for each animal. Values are expressed as mean +/− SEM (control, n=8; trauma, n=8). *Different from control, two-tailed t test (P < 0.05).

Figure 4

A. Cardiac MRI to measure left ventricular ejection fraction (LVEF) after TBI surgery (trauma) or sham surgery (control). Rats recovered from surgery for 2 days and were then placed under isofluorane anesthesia for respiratory and ECG-gated cardiac MRI. LVEF was estimated by determining the average ratio of biplane area of short-axis images of the ventricle in end-systole and end-diastole at three different LV sections (base, mid-LV, and apex) for each animal. Representative images from diastole and systole are shown with the LV cavity highlighted. B. Summary LVEF data. Values are expressed as mean +/− SEM (control, n=4; trauma, n=6). *Different from control, two-tailed t test (P < 0.05).

Figure 5

A. Reactive oxygen species (ROS) in LV myocardium after sham surgery (control, n=12), TBI surgery (trauma, n=12), or TBI surgery followed by treatment with propranolol (trauma + propranolol, n=12). Rats were euthanized 7– 10 days after surgery for acquisition of myocardial tissue and fresh frozen sections were incubated with dihydroethidium (DHE) to determine levels of ROS detected by fluorescence using Zeiss LSM 510 META Laser Scanning Microscope. Negative control slides for each animal were incubated with super oxide dismutase (SOD) immediately prior to addition of DHE (trauma + SOD, n=9). Representative images are shown. B. Graph depicts DHE fluorescence from myocardial tissue sections. Values are expressed as mean +/− SEM *Different from control, ANOVA (P < 0.05).