Influence of different fixation procedures on the quantification of infarction and oedema in a rat model of stroke

In pharmacodynamic studies using focal ischaemia models, the size of the in farct measured by quantitative histology is the most important outcome meas ure. Precise, unbiased and reproducible assessment of infarct volume is of foremost importance. A frequent problem in interventional stroke models is the evaluation of infarcts in animals found dead, where instant post-mortem fixation of the brain cannot be performed. The purpose of this study was t o investigate possible bias from perfusion, immediate and 3-h post-mortem d elayed immersion fixation on the measured volumes of cerebral infarction, o edema and hemispheres in a rat embolic stroke model. Thirty-six male Spragu e-Dawley rats were thromboembolized into the internal carotid artery. After survival for 24 h, the animals were divided into three groups: group 1 - i mmediate perfusion fixation; group 2 - immediate immersion fixation of the brain; and group 3 - animals left dead for 3 h at room temperature before r emoval of the brain for immersion fixation. Following histological preparat ion and evaluation, the volumes of the hemispheres and infarction were meas ured by quantitative histology and planimetry. Brains fixed by immersion we re 7% larger than the perfusion-fixed brains. Delaying the immersion fixati on for 3 h may increase hemisphere volume by a further 12%. Independent of the fixation procedure, the size of infarction was similar to 40% of the ip silateral hemisphere, and the oedema was similar to 11% of the size of the infarct. The used planimetric technique was accurate with measured values w ithin +/- 2% of the factual value. In conclusion, sizes of hemispheres, inf arction and oedema in absolute volume measures are influenced by the effect of unwanted variation of brain size caused by biological factors and artif icial shrinkage caused by fixation, dehydration and heat treatment of the s pecimens. Infarction and oedema expressed relatively in per cent of hemisph ere and infarct, respectively, are robust measures independent of the inves tigated fixation procedures.