Bilirubin oxidation products seen post subarachnoid hemorrhage have greater effects on aged rat brain compared to young.
BACKGROUND
We have previously shown that novel oxidation products of Bilirubin, called Bilirubin oxidation products (BOXes), are found in humans and animal models post subarachnoid hemorrhage. We have also proposed that BOXes may play a role in the pathogenesis and clinical complications post SAH. In this study we report on the direct toxicity effects of BOXes on rat brain.


METHODS
Identical volumes of either vehicle (normal saline) or BOXes (30 μl of a 20 μM solution) were applied above the dura through a cranial window of young (approximately 7-13 weeks) and aged (approximately 12-18 months) adult male Sprague Dawley rats (Charles River, Wilmington, MA, USA). To determine the extent of BOX-mediated injury, histology and immunocytochemistry were performed at 1, 2, 4, and 7 days post-surgical application of BOXes. We assessed the area of stress gene induction of HSP25/27 and HSP32. Immunohistochemistry was performed using standard avidin-biotin techniques. A monoclonal antibody to HSP25/27 (StressGen, Victoria, British Columbia, Canada), a monoclonal antibody to HSP32/HO-1 (StressGen), and a polyclonal HSP 32/HO-1 antibody were used for the immunocytochemistry.


RESULTS
A single dose of BOXes produced substantial increases in HSP25 and HO-1 in the aged rats at all early time points (≤4 days). After 7 days all groups were not significantly different than saline control. Young rats were resistant to BOXes effects compared to saline control with trends towards increased stress gene expression caused by BOXes that did not reach statistical significance.


CONCLUSIONS
We conclude from these studies that BOXes have direct effects on stress gene expression of the cortex post single dose application and that this can be seen for several days with apparent resolution at about 7 days. If BOXes are produced at similar levels in patients, the latency and duration of some SAH complications are consistent with these results.
Department of Neurology, University of Cincinnati, 670536, Cincinnati, OH 45267-0536, USA.
Joseph F
JF
Clark
Amanda
A
Harm
Ashlie
A
Saffire
Susan J
SJ
Biehle
Aigang
A
Lu
Gail J
GJ
Pyne-Geithman
D000375:Aging; D000704:Analysis of Variance; D000818:Animals; D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism*; D001663:Bilirubin / Q000737:chemistry / Q000378:metabolism*; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D004195:Disease Models, Animal; D005786:Gene Expression Regulation / Q000187:drug effects; D055551:HSP27 Heat-Shock Proteins / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism; D051547:Heme Oxygenase-1 / Q000378:metabolism; D008297:Male; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D013053:Spectrophotometry / Q000379:methods; D013345:Subarachnoid Hemorrhage / Q000473:pathology*; D013997:Time Factors
D000975:Antioxidants; D055551:HSP27 Heat-Shock Proteins; C486114:Hspb1 protein, rat; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; C491523:Hmox1 protein, rat; D001663:Bilirubin