Early disruptions of the blood-brain barrier may contribute to exacerbated neuronal damage and prolonged functional recovery following stroke in aged rats

Abstract

We examined the effects of age on stroke progression and outcome in order to explore the association between blood-brain barrier (BBB) disruption, neuronal damage, and functional recovery. Using middle cerebral artery occlusion (MCAO), young (3 months) and aged (18 months) rats were assessed for BBB disruption at 20min post-MCAO, and 24h post-MCAO with tissue plasminogen activator induced reperfusion at 120min. Results showed that BBB disruptions in aged rats occurred early and increased nearly two-fold at both the 20min and 24h time points when compared to young animals. Neuronal damage in aged rats was increased two-fold as compared to young rats at 24h, while no neuronal damage was observed at 20min. Young and aged rats exhibited neurological deficits when compared to sham-controls out to 14 days following MCAO and reperfusion; however, aged rats exhibited more severe onset of deficits and prolonged recovery. Results indicate that aged rats suffer larger infarctions, reduced functional recovery and increased BBB disruption preceding observable neuronal injury.

Fig. 1

TTC stained sections representative of young (A) and aged animals (B) infarction distribution 24 h post-MCAO and rt-PA reperfusion. (C) Volumetric analysis of cerebral infarctions. Infarction volumes are expressed as a percentage of the contralateral structure±S.E. *Significant difference in 18 month vs. 3 month age group (P≤0.006). N= 10 for all groups.

Fig. 2

Representative photomicrographs of slices from young (A) and aged (B) animals, 24 h post-MCAO and rt-PA treatment. In the young rats (A), a central area of coagulation necrosis is evident (closed arrowhead). This area contains sparse aggregates of acute inflammatory cells (*) (PMNs). The margin of the infarct contains swollen neurons, some with vacuolated neuropil (arrow). In the aged animals (B), the area of coagulation necrosis (closed arrowhead) is partially obscured by dense inflammatory cell infiltration (*) and petechial hemorrhages (open arrowhead). Vacuolation of the neuropil is evident (arrow) (scale bar = 100 μm).

Fig. 3

Performance on functional tests performed from 1 to 14 days post-occlusion to access neurological deficit. (A) mNSS, A composite functional score, judged on a scale of 0-17 with higher scores correlating to increased severity of functional deficits (N= 10/group). (B) Bracing test reflects number of postural adjustments made by the animals while being pushed laterally toward the paretic side over 90 cm (N= 5/group). (C) Placing test reflects the number of directed paw placements made in response to somatomotor stimulus over 10 trials (N= 5/group). (D) Tactile removal test reflects the time taken for animal to remove somatosensory stimulus placed on distal forearm (N= 5/group). (E) Akinesia test reflects the number of steps made with usable forearm over 30 s trial (N= 5/group). Scores are means±S.E.

Fig. 4

(A) Extravasation of albumin across the BBB assessed by quantification of Evan’s blue dye 24 h post-MCAO. Aged group untreated and treated with rt-PA exhibited greater BBB permeability in relation to corresponding young animals and contralateral hemisphere (*P < 0.01) (N= 5). (B) Photomicrographs of Fluoro-Jade B stained sections from a young (frame 1) and aged (frame 2) animal 24 h post-MCAO and rt-PA reperfusion. Damaged neurons within the infarcted region are fluorescently stained (green), while surviving neurons within the pneumbra are left unstained (scale bar = 100 μm). Frames 3-6, Confocal microscopy of vascular permeability markers dextran (70 kDa) and fibrinogen (300 kDa) 24 h post-MCAO and rt-PA reperfusion, taken in the penumbral region of the infarct. Frames 3 and 4, Confocal images of fibrinogen (red) and dextran (green) reactivity in the ipsilateral cortex of young (frame 3) and aged (frame 4) animals. No dextran extravasation is observed in the young animal (frame 3) and is similar to images from contralateral hemisphere of the aged animal (frame 5). Dextran is present in the extracellular space of aged (frame 4) animals at the same time point. Frame 6, Image in bright field that depicts lumen of cerebral microvessel. Fibrinogen can be seen remaining within the lumen of the vessel, while dextran reactivity is present along the outer surface in an aged animal that underwent MCAO and rt-PA reperfusion (scale bar = 5 μm).

Fig. 5

(A) Extravasation of albumin across the BBB assessed by quantification of Evan’s blue dye 20 min post-MCAO. Aged group exhibited greater BBB permeability in relation to corresponding young animals and the contralateral hemisphere (*) (P < 0.001). (B) Photomicrographs of Fluoro-Jade B stained section from a young (frame 1) and aged (frame 2) animal 20 min post-MCAO. Damaged neurons were not observed within the MCA perfusion territory in either young or aged animals (scale bar = 100 μm). Frames 3 and 4, Confocal images of fibrinogen (red) and dextran (green) reactivity in the ipsilateral cortex of young (frame 3) and aged (frame 4) animals, taken in the penumbral region of the infarct. No dextran extravasation is appreciated in the young rats (3) and is similar to images from contralateral hemisphere of the aged rats (not shown). Dextran is present in the extracellular space of aged (4) animals at the same time point (scale bar = 5 μm).