Methods for detection of brain injury after photothrombosis-induced ischemia in mice: Characteristics and new aspects of their application

Abstract

Background: Photothrombosis is a minimally invasive method for induction of cortical ischemia. However, different ways of applying some methods to assess photothrombosis-induced damage need to be developed.

New methods: We applied the tongue protrusion test and H&E staining of brain sections to detect ischemic damage after photothrombosis. Evaluation of the local status of the BBB using Evans blue dye was proposed. We also assessed the sensitivity of the grid-walk test. Moreover, we examined the interchangeability of MRI and TTC staining to measure lesion volume.

Results: We evaluated ischemic outcomes at 24 h after photothrombosis in mice. The tongue protrusion test did not reveal impairments of the neurological status whereas the grid-walk test showed the high sensitivity. Using histological techniques, we determined the reduction in the number of neurons with normal morphology in the penumbra. 3D reconstruction of the brain, which reflected Evans blue dye distribution in the nervous tissue, revealed BBB disruption in areas remote from the ischemic core. We also showed the strong correlation between damage volumes assessed by MRI and TTC staining.

Comparison with existing methods: The present work demonstrates the efficacy of the classical histological approach and TTC staining that are more affordable than MRI and immunohistochemical methods. Detection of 3D distribution of Evans blue dye in the brain in contrast to its total extraction reveals BBB damage in details.

Conclusions: We proposed the simple methods for describing the severity of brain ischemia at the cellular and whole organism levels without significant labor and financial expenditures.

Keywords: Hematoxylin and eosin staining; Ischemic injury volume; Local status of blood-brain barrier; Photothrombosis-induced focal ischemia; Sensorimotor tests.