In a rat model of transient ischemia,acute albumin leakage across the blood-brain barrier is often followed by rapid cellular uptake

Introduction

Evans blue-tagged albumin (EBA) is often used as a marker of blood-brain barrier (BBB) opening. Following cold injury and transient ischemia, EBA crosses the BBB and is taken up by neurons, and DNA fragmentation occurs within such neurons ¹ . In view of this, it was hypothesized and tested in a model of transient ischemia and subsequent hemorrhagic transformation (HT) that EBA crosses the leaky BBB and is rapidly taken up by brain cells during the acute phase of reperfusion.

Methods

In adult male Wistar rats (275–300 g), one middle cerebral artery (MCA) was occluded by suture for 3 hr. After suture withdrawal and 3 hr of reperfusion, EBA was intravenously injected. After 5 min (n=4) or 20 min (n=4), rats were sacrificed, and brains quickly removed and prepared for laser scanning confocal microscopy (LSCM). Three-dimensional LSCM reconstructions (30 μ thick) were made of EBA fluorescence in both sides of the brain at 5 sites within the preoptic area (POA), striatum (STR), and parietal cortex (CTX). The LSCM system was set to yield volumes of EBA fluorescence (Vf) on the contralateral side equal to the normal radiolabeled albumin distribution volume ² . At that setting, Vf was determined on the ischemic side and expressed as volume percent (%). The interstitial, neuronal and microglial distribution of EBA was evaluated in all 3D reconstructions.

Results

Normal capillary EBA distribution patterns were seen in 39 of the 40 reconstructions from the ipsilateral CTX; some EBA leakage and neuronal uptake was noted in one case. The ipsilateral side data for the STR were similar to those of the CTX. In the ipsilateral POA, however, EBA leakage (BBB opening) was detected in 31 of the 40 POA reconstructions. Cellular uptake of EBA was evident in the ipsilateral POA in all animals of the 5 min group. Among the 40 reconstructions from both time groups, 25 showed neuronal and 24, microglial uptake of EBA. For the POA, Vf (mean±SD) was 0.05±0.2% on the contralateral side and 2.7±1.5% and 13.7±13.7 on the ipsilateral side at 5 and 20 min, respectively. The large SD values indicate the great variability in EBA distribution among the 40 ischemic side reconstructions.

Conclusions

Supporting the hypothesis, appreciable uptake of leaked EBA by neurons and microglia occurred by 5 min in the POA. The lack of EBA (MW=68,000 Da) leakage in the STR was striking. In this model, much smaller MR contrast agent Gd-DTPA (MW=551 Da) and sucrose (342 Da) both pass across the BBB within the STR ³ . At this stage, opening of the BBB in STR, and possibly CTX, may only be to smaller molecules and the neurons and microglia are “protected” from albumin uptake by a semi-intact BBB. Tightening the BBB in such areas may be a way to prevent further damage and progression to HT.