Hematoma and perihematomal regions after intracerebral hemorrhage (ICH) are biochemically active environments known to undergo potent oxidizing reactions. We report facile production of bilirubin oxidation products (BOXes) via hemoglobin/Fenton reaction under conditions approximating putative in vivo conditions seen following ICH. Using a mixture of human hemoglobin, physiological buffers, unconjugated solubilized bilirubin, and molecular oxygen and/or hydrogen peroxide, we generated BOXes, confirmed by spectral signature consistent with known BOXes mixtures produced by independent chemical synthesis, as well as HPLC-MS of BOX A and BOX B. Kinetics are straightforward and uncomplicated, having initial rates around 0.002 microM bilirubin per microM hemoglobin per second under normal experimental conditions. In hematomas from porcine ICH model, we observed significant production of BOXes, malondialdehyde, and superoxide dismutase, indicating a potent oxidizing environment. BOX concentrations increased from 0.084 +/- 0.01 in fresh blood to 22.24 +/- 4.28 in hematoma at 72h, and were 11.22 +/- 1.90 in adjacent white matter (nmol/g). Similar chemical and analytical results are seen in ICH in vivo, indicating the hematoma is undergoing similar potent oxidations. This is the first report of BOXes production using a well-defined biological reaction and in vivo model of same. Following ICH, amounts of unconjugated bilirubin in hematoma can be substantial, as can levels of iron and hemoglobin. Oxidation of unconjugated bilirubin to yield bioactive molecules, such as BOXes, is an important discovery, expanding the role of bilirubin in pathological processes seen after ICH.
Intracerebral hemorrhage (ICH) and traumatic brain injury can induce brain tissue edema (i.e., interstitial and/or vasogenic), containing high concentrations of plasma proteins. To understand biochemical processes in edema development following these insults, it would be useful to examine alterations in various proteins (e.g., transcription factors, signaling). However, determining altered protein responses in edematous brain tissue using standard immunoblotting techniques is problematic due to contaminating plasma proteins. To solve this problem, we developed an enzyme-linked immunosorbent assay (ELISA) method to quantify the two major plasma proteins, albumin and immunoglobulin G (IgG), that comprise about 80% of the total plasma proteins. We tested our method on edematous white matter samples from our porcine ICH model. To induce ICH, we infused autologous arterial whole blood (3 mL) into frontal hemispheric white matter of pentobarbital- anesthetized pigs ( approximately 20 kg) over 15 min. We froze brains in situ at various times up to 24 h post- ICH and sampled white matter adjacent and contralateral to hematomas. We prepared cytoplasmic extracts that we subjected to ELISA and immunoblotting analyses. Our results demonstrate that this ELISA method is accurate, reproducible, and enables the concentrations of albumin and IgG in edematous brain tissue samples to be accurately determined. By using this correction method, equal amounts of cellular protein can be loaded onto gels during immunoblotting procedures. This method is applicable to edematous tissue samples in brain injury models in which high plasma protein concentrations result from interstitial or vasogenic edema development.
Ultrasound acts synergistically with thrombolytic agents, such as recombinant tissue plasminogen activator (rt-PA), to accelerate thrombolysis. The aim of the study was to demonstrate the efficacy of 120-kHz ultrasound-enhanced rt-PA thrombolysis in a porcine hemorrhagic stroke model in vivo. Clots were formed by infusing 3 ml of autologous blood into the frontal white matter of 30 mixed-bred Yorkshire pigs (20.5–3.1 kg) and incubated for 3 h. For these nonsurvival studies, six pigs received rt-PA alone (0.3 cc of 0.107 mg/ml), six received ultrasound alone, six received rt-PA plus ultrasound, six were sham-exposed (saline only), and six were controls (no ultrasound or rt-PA treatment). The clots receiving ultrasound treatment were insonified with a peak-to-peak pressure of 0.48 MPa in situ (80% duty cycle, and PRF of 1.7 kHz) for 30 min. Clots treated with rt-PA alone exhibited a volume loss of 55.0% and clots treated with rt-PA and 120-kHz ultrasound had a significantly higher volume loss of 75.2% and a higher penetration of rt-PA. Thus, 120-kHz pulsed ultrasound enhancement of thrombolysis has been demonstrated both in vitro and in an in vivo porcine hemorrhagic stroke model.
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