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

Abstract

In pharmacodynamic studies using focal ischaemia models, the size of the infarct measured by quantitative histology is the most important outcome measure. 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 to investigate possible bias from perfusion, immediate and 3-h post-mortem delayed immersion fixation on the measured volumes of cerebral infarction, oedema and hemispheres in a rat embolic stroke model. Thirty-six male Sprague-Dawley rats were thromboembolized into the internal carotid artery. After survival for 24 h, the animals were divided into three groups: group 1 - immediate perfusion fixation; group 2 - immediate immersion fixation of the brain; and group 3 - animals left dead for 3 h at room temperature before removal of the brain for immersion fixation. Following histological preparation and evaluation, the volumes of the hemispheres and infarction were measured by quantitative histology and planimetry. Brains fixed by immersion were 7% larger than the perfusion-fixed brains. Delaying the immersion fixation for 3 h may increase hemisphere volume by a further 12%. Independent of the fixation procedure, the size of infarction was approximately 40% of the ipsilateral hemisphere, and the oedema was approximately 11% of the size of the infarct. The used planimetric technique was accurate with measured values within +/- 2% of the factual value. In conclusion, sizes of hemispheres, infarction and oedema in absolute volume measures are influenced by the effect of unwanted variation of brain size caused by biological factors and artificial shrinkage caused by fixation, dehydration and heat treatment of the specimens. Infarction and oedema expressed relatively in per cent of hemisphere and infarct, respectively, are robust measures independent of the investigated fixation procedures.

The site of embolization related to infarct size, oedema and clinical outcome in a rat stroke model - further translational stroke research

“…The 3 different fixation procedures were evenly distributed without significant differences between the 3 groups with different grades of vascular occlusion. As shown previously, fixation procedures had no influence on the results of this current investigation, because infarct and oedema volumes calculated as percentages of the corresponding hemisphere were independent of fixation procedures [6]. …”

“…Animals can die from herniation caused by large hemispheric infarcts and ipsilateral brain oedema within 24-48 hours [29]. As shown previously the three different fixation procedures did not interfere with the measurement of oedema, which after 24 hours constituted 11% of the infarct volume [6], compared to 6% using comparable methods [28], provided all enlargement of the right hemisphere was located inside the infarction, which may not be absolutely valid [30]. The calculations reported by Leach et al [31] and Swanson et al [32] compensate for these problems, and the volume effect of oedema is attempted weighted out.…”

“…Animals in this study have previously been investigated for other purposes [6]. Re-using animals to provide extended results and observations is in favour of the methodology encompassed in the concepts of refinement, reduction and replacement within animal laboratory science [7,8].…”

Multi-modal characterization of rapid anterior hippocampal volume increase associated with aerobic exercise

“…However, the disadvantage of histology is that only a small portion of a given region is sampled, whereas imaging allows the signal to be averaged across the entire region of issue. Histology also requires fixation which, depending on the precise procedure used, can cause shrinkage and dehydration, potentially distorting volume measurements (Overgaard and Meden, 2000). …”

Thinning, movement, and volume loss of residual cortical tissue occurs after stroke in the adult rat as identified by histological and magnetic resonance imaging analysis

“…This presents a problem however, as the histological preparation of neural tissue is known to introduce a number of morphological anomalies. Tissue perfusion, removal, staining, and dehydration can lead to artificial tissue shrinkage, nicks in surface brain areas, and changes in tissue shape and location (Garman, 1990;Overgaard and Meden, 2000;Sadowski et al, 1995;Simmons and Swanson, 2009;Skoglund et al, 1996). It is possible that any cortical thinning, movement, volume, or density loss observed after stroke in the adult rat could be exaggerated by histological preparation.…”