Severe blood-brain barrier disruption and surrounding tissue injury.
OBJECTIVE
Blood-brain barrier opening during ischemia follows a biphasic time course, may be partially reversible, and allows plasma constituents to enter brain and possibly damage cells. In contrast, severe vascular disruption after ischemia is unlikely to be reversible and allows even further extravasation of potentially harmful plasma constituents. We sought to use simple fluorescent tracers to allow wide-scale visualization of severely damaged vessels and determine whether such vascular disruption colocalized with regions of severe parenchymal injury.


METHODS
Severe vascular disruption and ischemic injury was produced in adult Sprague Dawley rats by transient occlusion of the middle cerebral artery for 1, 2, 4, or 8 hours, followed by 30 minutes of reperfusion. Fluorescein isothiocyanate-dextran (2 MDa) was injected intravenously before occlusion. After perfusion-fixation, brain sections were processed for ultrastructure or fluorescence imaging. We identified early evidence of tissue damage with Fluoro-Jade staining of dying cells.


RESULTS
With increasing ischemia duration, greater quantities of high molecular weight dextran-fluorescein isothiocyanate invaded and marked ischemic regions in a characteristic pattern, appearing first in the medial striatum, spreading to the lateral striatum, and finally involving cortex; maximal injury was seen in the mid-parietal areas, consistent with the known ischemic zone in this model. The regional distribution of the severe vascular disruption correlated with the distribution of 24-hour 2,3,5-triphenyltetrazolium chloride pallor (r=0.75; P<0.05) and the cell death marker Fluoro-Jade (r=0.86; P<0.05). Ultrastructural examination showed significantly increased areas of swollen astrocytic foot process and swollen mitochondria in regions of high compared to low leakage, and compared to contralateral homologous regions (ANOVA P<0.01). Dextran extravasation into the basement membrane and surrounding tissue increased significantly from 2 to 8 hours of occlusion duration (Independent samples t test, P<0.05).


CONCLUSIONS
Severe vascular disruption, as labeled with high-molecular-weight dextran-fluorescein isothiocyanate leakage, is associated with severe tissue injury. This marker of severe vascular disruption may be useful in further studies of the pathoanatomic mechanisms of vascular disruption-mediated tissue injury.
Chen
Bo
B
Department of Neurosciences, University of California San Diego, School of Medicine, La Jolla, USA.
Friedman
Beth
B
Cheng
Qun
Q
Tsai
Phil
P
Schim
Erica
E
Kleinfeld
David
D
Lyden
Patrick D
PD
D000818:Animals; D001812:Blood-Brain Barrier / Q000503:physiopathology*; D001921:Brain / Q000098:blood supply / Q000473:pathology / Q000503:physiopathology; D001929:Brain Edema / Q000209:etiology / Q000503:physiopathology*; D002545:Brain Ischemia / Q000150:complications / Q000473:pathology / Q000503:physiopathology*; D002536:Cerebral Arteries / Q000473:pathology / Q000503:physiopathology*; D003911:Dextrans / Q000493:pharmacokinetics; D004195:Disease Models, Animal; D018450:Disease Progression; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D005452:Fluoresceins; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D007202:Indicators and Reagents; D020244:Infarction, Middle Cerebral Artery / Q000150:complications / Q000473:pathology / Q000503:physiopathology*; D008297:Male; D009410:Nerve Degeneration / Q000209:etiology / Q000473:pathology / Q000503:physiopathology; D009930:Organic Chemicals; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling; D013778:Tetrazolium Salts
D003911:Dextrans; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009930:Organic Chemicals; D013778:Tetrazolium Salts; C015219:fluorescein isothiocyanate dextran; C435731:fluoro jade; D016650:Fluorescein-5-isothiocyanate