A Novel Method for Assessing Cerebral Edema, Infarcted Zone and Blood-Brain Barrier Breakdown in a Single Post-stroke Rodent Brain

Abstract

Stroke is a major cause of global morbidity and mortality. Middle cerebral artery occlusion (MCAO) has historically been the most common animal model of simulating ischemic stroke. The extent of neurological injury after MCAO is typically measured by cerebral edema, infarct zone, and blood-brain barrier (BBB) permeability. A significant limitation of these methods is that separate sets of brains must be used for each measurement. Here we examine an alternative method of measuring cerebral edema, infarct zone and BBB permeability following MCAO in the same set of brain samples. Ninety-six rats were randomly divided into three experimental groups. Group 1 (n = 27) was used for the evaluation of infarct zone and brain edema in rats post-MCAO (n = 17) vs. sham-operated controls (n = 10). Group 2 (n = 27) was used for the evaluation of BBB breakdown in rats post-MCAO (n = 15) vs. sham-operated controls (n = 10). In Group 3 (n = 42), all three parameters were measured in the same set of brain slices in rats post-MCAO (n = 26) vs. sham-operated controls (n = 16). The effect of Evans blue on the accuracy of measuring infarct zone by 2,3,5-triphenyltetrazolium chloride (TTC) staining was determined by measuring infarct zone with and without an applied blue filter. The effects of various concentrations of TTC (0, 0.05, 0.35, 0.5, 1, and 2%) on the accuracy of measuring BBB permeability was also assessed. There was an increase in infarct volume (p < 0.01), brain edema (p < 0.01) and BBB breakdown (p < 0.01) in rats following MCAO compared to sham-operated controls, whether measured separately or together in the same set of brain samples. Evans blue had an effect on measuring infarct volume that was minimized by the application of a blue filter on scanned brain slices. There was no difference in the Evans blue extravasation index for the brain tissue samples without TTC compared to brain tissue samples incubated in TTC. Our results demonstrate that measuring cerebral edema, infarct zone and BBB permeability following MCAO can accurately be measured in the same set of brain samples.

Evaluation of the Effectiveness of Post-Stroke Metformin Treatment Using Permanent Middle Cerebral Artery Occlusion in Rats

“…The brain oedema area was assessed and calculated in arbitrary units (pixels) from the summation of coronal slice areas using ImageJ/Fiji 1.46 software. The brain oedema area was expressed as a percentage of the normal areas in the contralateral unaffected hemisphere [ 52 ].…”

A Novel Histological Technique to Assess Severity of Traumatic Brain Injury in Rodents: Comparisons to Neuroimaging and Neurological Outcomes

“…Brain edema, lesion volume, and BBB breakdown were measured in the same set of brain samples following a protocol described previously in a stroke model ( Kuts et al, 2019a ). 2% Evans blue in saline (4 ml/kg) was administered through a cannulated tail vein as a blood-brain permeability tracer and was allowed to circulate for 60 min.…”

“…We measured the brain edema in pixels, using ImageJ software ( Kuts et al, 2019a ). Brain edema was expressed as a percentage of the normal areas in the contralateral unaffected hemisphere.…”

“…Obtaining histological measurements of outcomes post-TBI in preclinical experimental models historically requires three separate groups of animals for the evaluation of BBB breakdown, brain edema, and lesion volume, respectively ( Başkaya et al, 2000 ; Meymandi et al, 2018 ; Soltani et al, 2018 ). In rodent models of stroke, methods that utilize multiple histological outcomes in the same brain set have been well described ( Li et al, 2018 ; Kuts et al, 2019a ; Sanchez Bezanilla et al, 2019 ). However, no such methods have been previously described in preclinical rodent models of TBI.…”

TMEM16F Aggravates Neuronal Loss by Mediating Microglial Phagocytosis of Neurons in a Rat Experimental Cerebral Ischemia and Reperfusion Model

“…Motor deficits were graded from 0 to 4. A score of 0 was given for no visible neurological deficits; a score of 1 was given for forelimb flexion; a score of 2 was given for contralateral weak forelimb grip (the operator places the animal on an absorbent pad and gently pulls the tail); a score of 3 was given for circling to the paretic side only when the tail was stimulated; and a score of 4 was given for spontaneous circling ( 20 , 22 , 23 ). Finally, animals with positive performances were included in our study.…”