A Comparative Study of Variables Influencing Ischemic Injury in the Longa and Koizumi Methods of Intraluminal Filament Middle Cerebral Artery Occlusion in Mice.

Gary P Morris,Amanda L Wright,Bryce Vissel,Lars M Ittner,Richard P Tan,Amadeus Gladbach,Lucio Annunziato

doi:10.1371/journal.pone.0148503

Abstract

The intraluminal filament model of middle cerebral artery occlusion (MCAO) in mice and rats has been plagued by inconsistency, owing in part to the multitude of variables requiring control. In this study we investigated the impact of several major variables on survival rate, lesion volume, neurological scores, cerebral blood flow (CBF) and body weight including filament width, time after reperfusion, occlusion time and the choice of surgical method. Using the Koizumi method, we found ischemic injury can be detected as early as 30 min after reperfusion, to a degree that is not statistically different from 24 h post-perfusion, using 2,3,5-Triphenyltetrazolium chloride (TTC) staining. We also found a distinct increase in total lesion volume with increasing occlusion time, with 30–45 min a critical time for the development of large, reproducible lesions. Furthermore, although we found no significant difference in total lesion volume generated by the Koizumi and Longa methods of MCAO, nor were survival rates appreciably different between the two at 4 h after reperfusion, the Longa method produces significantly greater reperfusion. Finally, we found no statistical evidence to support the exclusion of data from animals experiencing a CBF reduction of <70% in the MCA territory following MCAO, using laser-Doppler flowmetry. Instead we suggest the main usefulness of laser-Doppler flowmetry is for guiding filament placement and the identification of subarachnoid haemorrhages and premature reperfusion. In summary, this study provides detailed evaluation of the Koizumi method of intraluminal filament MCAO in mice and a direct comparison to the Longa method.

Highlights

The most common human focal cerebral ischemia occurs due to thrombotic or embolic occlusion of the middle cerebral artery (MCA)
Previous studies have shown that the volume of ischemic lesion grows between 0–24 h, peaking at 24 h, when assessed by Triphenyltetrazolium chloride (TTC) staining following the intraluminal filament model of MCA occlusion (MCAO) in mice [14]
Previous studies have suggested the tip width of intraluminal filaments used for MCAO can alter resulting volumes of ischemic lesions [30]

Summary

Introduction

The most common human focal cerebral ischemia occurs due to thrombotic or embolic occlusion of the middle cerebral artery (MCA). Rodent models have predominantly aimed to mimic MCA occlusion (MCAO), no single rodent model fully recapitulates the features of human MCAO and each model has various advantages and disadvantages [1,2]. Despite having been utilised for almost three decades, and many attempts to reduce variability [8,9], the intraluminal filament method still displays large inconsistency in the volume of ischemic lesions generated, both within studies and from lab to lab. Dirnagl highlighted standard deviations in infarct volumes are as much as 40% of the mean in many studies [10]. These figures are indicative of the difficulty in generating robust and reproducible data from pre-clinical stroke studies

Methods

Results

Discussion

Conclusion